Diagnostics and therapeutics for restenosis

ABSTRACT

Methods and kits for determining whether a subject has or is predisposed to developing restenosis are provided.

1. BACKGROUND OF THE INVENTION

Restenosis

Percutaneous transluminal coronary angioplasty (PTCA) is used to treatobstructive coronary artery disease by compressing atheromatous plaqueto the sides of the vessel wall. PTCA is widely used with an initialsuccess rate of over 90%. Approximately 666,000 angioplasties wereconducted in the United States alone in 1996, and more of theseprocedures were performed on men (452,000) than women (214,000). Of thistotal, 482,000 were percutaneous transluminal coronary angioplasty(P.T.C.A. (American Heart Association; www.amhrt.org). Despite thefrequent application of this procedure and its high initial successrate, the long-term success of PTCA is limited by intraluminalrenarrowing or restenosis at the site of the procedure. This occurswithin 6 months following the procedure in approximately 30% to 40% ofpatients who undergo a single vessel procedure and in more than 50% ofthose who undergo multivessel angioplasty.

Stent placement has largely supplanted balloon angioplasty because it isable to more widely restore intraluminal dimensions which has the effectof reducing restenosis by approximately 50%. Ironically, stent placementactually increases neointimal growth at the treatment site, but becausea larger lumen can be achieved with stent placement, the tissue growthis more readily accommodate, and sufficient luminal dimensions aremaintained, so that the restenosis rate is nearly halved by stentplacement compared with balloon angioplasty alone.

The pathophysiological mechanisms involved in restenosis are not fullyunderstood. While a number of clinical, anatomical and technical factorshave been linked to the development of restenosis, at least 50% of theprocess has yet to be explained. However, it is known that followingendothelial injury, a series of repair mechanisms are initiated. Withinminutes of the injury, a layer of platelets and fibrin is deposited overthe damaged endothelium. Within hours to days, inflammatory cells beginto infiltrate the injured area. Within 24 hours after an injury,vascular smooth muscle cells (SMCs) located in the vessel media commenceDNA synthesis. A few days later, these activated, synthetic SMCs migratethrough the internal elastic lamina towards the luminal surface. Aneointima is formed by these cells by their continued replication andtheir production of extracellular matrix. An increase in the intimalthickness occurs with ongoing cellular proliferation matrix deposition.When these processes of vascular healing progress excessively, thepathological condition is termed intimal hyperplasia or neointimialhyperplasia. Histological studies in animal models have identifiedneointimal hyperplasia as the central element in restenosis.

Neointimal hyperplasia is understood to figure prominently in peripheralvascular restenosis following reconstructive procedures. One series of5,000 arterial reconstructions reports 50% of late failures to be due toneointimal hyperplasia (Imparato et al. (1972) Surg. 72:1107-1117).Restenosis following stenting is similarly thought to involve animportant component of neointimal hyperplasia (Dussaillant et al. (1995)J. Am. Coll. Cardiol 26:720-724). In the coronary system, by contrast,restenosis following balloon angioplasty involves vascular remodeling aswell as neointimal hyperplasia. The importance of vascular remodeling inthis setting may be attributable to the nature of the injury to thevessel wall following balloon angioplasty. Commonly, the injury to thevessel wall with this procedure involves dissection planes extendingthrough the atherosclerotic plaque into the vessel media (Mintz et al.(1996) Circ. 94:35043). Furthermore, plaque fracture, medial stretch,focal medial rupture and adventitial stretch all may occur followingangioplasty. Repair of the deeper layers of the vessel wall takes placeby the general processes of wound healing, including inflammation,neovascularization, fibroblast proliferation and eventual collagendeposition. Cumulatively, these processes lead to remodeling of thecoronary vessel wall that may culminate in restenosis.

The biology of vascular wall healing implicated in restenosis thereforeincludes the general processes of wound healing and the specificprocesses of neointimal hyperplasia. Inflammation is generally regardedas an important component in both these processes. (Munro and Cotran(1993) Lab. Investig. 58:249-261; and Badimon et al. (1993), Supp II87:3-6). Understanding the effects of acute and chronic inflammation inthe blood vessel wall can thus suggest methods for diagnosing andtreating restenosis and related conditions.

In its initial phase, inflammation is characterized by the adherence ofleukocytes to the vessel wall. Leukocyte adhesion to the surface ofdamaged endothelium is mediated by several complex glycoproteins on theendothelial and neutrophil surfaces. Two of these binding molecules havebeen well-characterized: the endothelial leukocyte adhesion molecule-1(ELAM-1) and the intercellular adhesion molecule-1 (ICAM-1). Duringinflammatory states, the attachment of neutrophils to the involved cellsurfaces is greatly increased, primarily due to the upregulation andenhanced expression of these binding molecules. Substances thought to beprimary mediators of the inflammatory response to tissue injury,including interleukin-1 (IL-1), tumor necrosis factor alpha (TNF-α),lymphotoxin and bacterial endotoxins, all increase the production ofthese binding substances.

After binding to the damaged vessel wall, leukocytes migrate into it.Once in place within the vessel wall, the leukocytes, in particularactivated macrophages, then release additional inflammatory mediators,including IL-1, TNF, prostaglandin E₂, (PGE₂), bFGF, and transforminggrowth factors α and β (TGFα, TGFβ). All of these inflammatory mediatorsrecruit more inflammatory cells to the damaged area, and regulate thefurther proliferation and migration of smooth muscle. A well-knowngrowth factor elaborated by the monocyte-macrophage is monocyte- andmacrophage-derived growth factor (MDGF), a stimulant of smooth musclecell and fibroblast proliferation. MDGF is understood to be similar toplatelet-derived growth factor (PDGF); in fact, the two substances maybe identical. By stimulating smooth muscle cell proliferation,inflammation can contribute to the development and the progression ofneointimal hyperplasia.

Leukocytes, attracted to the vessel wall by the abovementioned chemicalmediators of inflammation, produce substances that have direct effectson the vessel wall that may exacerbate the local injury and prolong thehealing response. First, leukocytes activated by the processes ofinflammation secrete lysosomal enzymes that can digest collagen andother structural proteins. Releasing these enzymes within the vesselwall can affect the integrity of its extracellular matrix, permittingSMCs and other migratory cells to pass through the wall more readily.Hence, the release of these lysosomal proteases can enhance theprocesses leading to neointimal hyperplasia. Second, activatedleukocytes produce free radicals by the action of the NADPH system ontheir cell membranes. These free radicals can damage cellular elementsdirectly, leading to an extension of a local injury or a prolongation ofthe cycle of injury-inflammation-healing.

The responses to vascular injury that lead to restenosis have certainfeatures in common with the processes leading to the development of thevascular lesions of atherosclerosis. Currently, it is understood thatthe lesions of atherosclerosis are initiated by some form of injury toarterial endothelium, whether due to hemodynarnic factors, endothelialdysfunction or a combination of these or other factors (Schoen, “Bloodvessels,” pp. 467-516 in Pathological Basis of Disease (Philadelphia:Saunders, 1994)). Inflammation has been implicated in the formation andprogression of atherosclerotic lesions. Several inflammatory products,including IL-1β, have been identified in atherosclerotic lesions or inthe endothelium of diseased coronary arteries (Galea, et al. (1996)Arterioscler Thromb Vasc Biol. 16:1000-6). Also, serum concentrations ofIL-1β are elevated in patients with coronary disease (Hasdai, et al.(1996) Heart, 76:24-8). Realizing the importance of inflammatoryprocesses in the final common pathways of vascular response to injuryallows analogies to be drawn between the lesions seen in restenosis andthose seen in atherosclerosis.

Currently, approximately 500,000 patients per year undergo vascularreconstructive procedures, with half involving the coronary vessels andthe other half involving the periphery. Restenosis and progressiveatherosclerosis are the most common mechanisms for late failure in thesereconstructions. It would be desirable to determine which patients wouldrespond well to invasive treatments for occlusive vascular disease suchas angioplasty and intravascular stent placement. It would be furtherdesirable to identify those patients at increased risk for stenosis sothat they could be targeted with appropriate therapies to prevent,modulate or reverse the condition. It would be desirable, moreover, toidentify those individuals for whom PTCA and stent placement is asuboptimal therapeutic choice because of the risk of restenosis. Thosepatients might become candidates at earlier stages for vascularreconstructive procedures, possibly combined with other pharmacologicalinterventions.

Genetics of the IL-1 Gene Cluster

The IL-1 gene cluster is on the long arm of chromosome 2 (2q13) andcontains at least the genes for IL-1α (IL-1A), IL-1β (IL-1B), and theIL-1 receptor antagonist (IL-1RN), within a region of 430 Kb (Nicklin,et al. (1994) Genomics, 19: 382-4). The agonist molecules, IL-1α andIL-1β, have potent pro-inflammatory activity and are at the head of manyinflammatory cascades. Their actions, often via the induction of othercytokines such as IL-6 and IL-8, lead to activation and recruitment ofleukocytes into damaged tissue, local production of vasoactive agents,fever response in the brain and hepatic acute phase response. All threeIL-1 molecules bind to type I and to type II IL-1 receptors, but onlythe type I receptor transduces a signal to the interior of the cell. Incontrast, the type II receptor is shed from the cell membrane and actsas a decoy receptor. The receptor antagonist and the type II receptor,therefore, are both anti-inflammatory in their actions.

Inappropriate production of IL-1 plays a central role in the pathologyof many autoimmune and inflammatory diseases, including rheumatoidarthritis, inflammatory bowel disorder, psoriasis, and the like. Inaddition, there are stable inter-individual differences in the rates ofproduction of IL-1, and some of this variation may be accounted for bygenetic differences at IL-1 gene loci. Thus, the IL-1 genes arereasonable candidates for determining part of the genetic susceptibilityto inflammatory diseases, most of which have a multifactorial etiologywith a polygenic component. Indeed, there is increasing evidence thatcertain alleles of the IL-1 genes are over-represented in thesediseases.

Certain alleles from the IL-1 gene cluster are already known to beassociated with particular disease states. For example, IL-1RN allele 2has been shown to be associated with coronary artery disease(PCT/US/98/04725, and U.S. Ser. No. 08/813,456), osteoporosis (U.S. Pat.No. 5,698,399), nephropathy in diabetes mellitus (Blakemore, et al.(1996) Hum. Genet. 97(3): 369-74), alopecia areata (Cork, et al., (1995)J. Invest. Dermatol. 104(5 Supp.): 15S-16S; Cork et al. (1996) DermatolClin 14: 671-8), Graves disease (Blakemore, et al. (1995) J. Clin.Endocrinol. 80(1): 111-5), systemic lupus erythematosus (Blakemore, etal. (1994) Arthritis Rheum. 37: 1380-85), lichen sclerosis (Clay, et al.(1994) Hum. Genet. 94: 407-10), and ulcerative colitis (Mansfield, etal. (1994) Gastoenterol. 106(3): 637-42).

In addition, the IL-1A allele 2 from marker −889 and IL-1B (TaqI) allele2 from marker +3954 have been found to be associated with periodontaldisease (U.S. Pat. No. 5,686,246; Kornrnan and diGiovine (1998) AnnPeriodont 3: 327-38; Hart and Kornman (1997) Periodontol 2000 14:202-15; Newman (1997) Compend Contin Educ Dent 18: 881-4; Kornman et al.(1997) J. Clin Periodontol 24: 72-77). The IL-1A allele 2 from marker−889 has also been found to be associated with juvenile chronicarthritis, particularly chronic iridocyclitis (McDowell, et al. (1995)Arthritis Rheum. 38: 221-28). The IL-1B (TaqI) allele 2 from marker+3954 of IL-1B has also been found to be associated with psoriasis andinsulin dependent diabetes in DR3/4 patients (di Giovine, et al. (1995)Cytokine 7: 606; Pociot, et al. (1992) Eur J. Clin. Invest. 22:396-402). Additionally, the IL-1RN (VNTR) allele 1 has been found to beassociated with diabetic retinopathy (see U.S. Ser. No. 09/037,472, andPCT/GB97/02790). Furthermore allele 2 of IL-1RN (VNTR) has been found tobe associated with ulcerative colitis in Caucasian populations fromNorth America and Europe (Mansfield, J. et al., (1994) Gastroenterology106: 637-42). Interestingly, this association is particularly strongwithin populations of ethnically related Ashkenazi Jews (PCTWO97/25445).

Genotype Screening

Traditional methods for the screening of heritable diseases havedepended on either the identification of abnormal gene products (e.g.,sickle cell anemia) or an abnormal phenotype (e.g., mental retardation).These methods are of limited utility for heritable diseases with lateonset and no easily identifiable phenotypes such as, for example, apredisposition to restenosis. With the development of simple andinexpensive genetic screening methodology, it is now possible toidentify polymorphisms that indicate a propensity to develop disease,even when the disease is of polygenic origin. The number of diseasesthat can be screened by molecular biological methods continues to growwith increased understanding of the genetic basis of multifactorialdisorders.

Genetic screening (also called genotyping or molecular screening), canbe broadly defined as testing to determine if a patient has mutations(or alleles or polymorphisms) that either cause a disease state or are“linked” to the mutation causing a disease state. Linkage refers to thephenomenon that DNA sequences which are close together in the genomehave a tendency to be inherited together. Two sequences may be linkedbecause of some selective advantage of co-inheritance. More typically,however, two polymorphic sequences are co-inherited because of therelative infrequency with which meiotic recombination events occurwithin the region between the two polymorphisms. The co-inheritedpolymorphic alleles are said to be in linkage disequilibrium with oneanother because, in a given human population, they tend to either bothoccur together or else not occur at all in any particular member of thepopulation. Indeed, where multiple polymorphisms in a given chromosomalregion are found to be in linkage disequilibrium with one another, theydefine a quasi-stable genetic “haplotype.” In contrast, recombinationevents occurring between two polymorphic loci cause them to becomeseparated onto distinct homologous chromosomes. If meiotic recombinationbetween two physically linked polymorphisms occurs frequently enough,the two polymorphisms will appear to segregate independently and aresaid to be in linkage equilibrium.

While the frequency of meiotic recombination between two markers isgenerally proportional to the physical distance between them on thechromosome, the occurrence of “hot spots” as well as regions ofrepressed chromosomal recombination can result in discrepancies betweenthe physical and recombinational distance between two markers. Thus, incertain chromosomal regions, multiple polymorphic loci spanning a broadchromosomal domain may be in linkage disequilibrium with one another,and thereby define a broad-spanning genetic haplotype. Furthermore,where a disease-causing mutation is found within or in linkage with thishaplotype, one or more polymorphic alleles of the haplotype can be usedas a diagnostic or prognostic indicator of the likelihood of developingthe disease. This association between otherwise benign polymorphisms anda disease-causing polymorphism occurs if the disease mutation arose inthe recent past, so that sufficient time has not elapsed for equilibriumto be achieved through recombination events. Therefore identification ofa human haplotype which spans or is linked to a disease-causingmutational change, serves as a predictive measure of an individual'slikelihood of having inherited that disease-causing mutation.Importantly, such prognostic or diagnostic procedures can be utilizedwithout necessitating the identification and isolation of the actualdisease-causing lesion. This is significant because the precisedetermination of the molecular defect involved in a disease process canbe difficult and laborious, especially in the case of multifactorialdiseases such as inflammatory disorders.

Indeed, the statistical correlation between an inflammatory disorder andan IL-1 polymorphism does not necessarily indicate that the polymorphismdirectly causes the disorder. Rather the correlated polymorphism may bea benign allelic variant which is linked to (i.e. in linkagedisequilibrium with) a disorder-causing mutation which has occurred inthe recent human evolutionary past, so that sufficient time has notelapsed for equilibrium to be achieved through recombination events inthe intervening chromosomal segment. Thus, for the purposes ofdiagnostic and prognostic assays for a particular disease, detection ofa polymorphic allele associated with that disease can be utilizedwithout consideration of whether the polymorphism is directly involvedin the etiology of the disease. Furthermore, where a given benignpolymorphic locus is in linkage disequilibrium with an apparentdisease-causing polymorphic locus, still other polymorphic loci whichare in linkage disequilibrium with the benign polymorphic locus are alsolikely to be in linkage disequilibrium with the disease-causingpolymorphic locus. Thus these other polymorphic loci will also beprognostic or diagnostic of the likelihood of having inherited thedisease-causing polymorphic locus. Indeed, a broad-spanning humanhaplotype (describing the typical pattern of co-inheritance of allelesof a set of linked polymorphic markers) can be targeted for diagnosticpurposes once an association has been drawn between a particular diseaseor condition and a corresponding human haplotype. Thus, thedetermination of an individual's likelihood for developing a particulardisease of condition can be made by characterizing one or moredisease-associated polymorphic alleles (or even one or moredisease-associated haplotypes) without necessarily determining orcharacterizing the causative genetic variation.

2. SUMMARY OF THE INVENTION

In one aspect, the present invention provides novel methods and kits fordetermining whether a subject has or is predisposed to developingrestenosis. Diagnosis of the presence of a restenosis disorderidentifies those patients predisposed to the development of a restenosisdisease, characterized by clinical events related to the recurrence ofthe initial vascular stenosis that is being treated by the stent.Determining which patients are at risk for developing the diseasebecause they have the disorder thus opens the possibility of selectingtherapies for the initial vascular stenosis most likely to avoidsubsequent stenoses. Such patients might be preferred candidates forsurgical revascularization rather than percutaneous transluminalangioplasty, for example, or such patients may benefit frompharmacological or topical interventions at an early stage that couldaffect the progression of the restenosis disorder.

In one embodiment, the method comprises determining whether a restenosisassociated allele is present in a nucleic acid sample obtained from thesubject. In a preferred embodiment, the restenosis associated allele isselected from the group consisting of allele 1 of each of the followingmarkers: IL-1A (+4845), IL-1B (+3954), IL-1B (−511), IL-1RN (+2018) andIL-1RN (VNTR) or an allele that is in linkage disequilibrium with one ofthe aforementioned alleles. In preferred embodiments, the presence of aparticular allelic pattern of one or more of the abovementioned IL-1polymorphic loci is used to predict the susceptibility of an individualto developing restenosis. In particular, there are three patterns ofalleles at four polymorphic loci in the IL-1 gene cluster that showvarious associations with particular cardiovascular disorders. Thesepatterns are referred to herein as patterns 1, 2 and 3. Pattern 1comprises an allelic pattern including allele 2 of IL-1A (+4845) orIL-1B (+3954) and allele 1 of IL-1B (−511) or IL-1RN (+2018), or anallele that is in linkage disequilibrium with one of the aforementionedallele. In a preferred embodiment, this allelic pattern permits thediagnosis of occlusive cardiovascular disorder. Pattern 2 comprises anallelic pattern including allele 2 of IL-1B (−511) or IL-1RN (+2018) andallele 1 of IL-1A (+4845) or IL-1B (+3954), or an allele that is inlinkage disequilibrium with one of the aforementioned alleles. In apreferred embodiment, this allelic pattern permits the diagnosis ofocclusive cardiovascular disorder. Pattern 3 comprises an allelicpattern including allele 1 of IL-1A (+4845) or allele 1 of IL-1B(+3954), and allele 1 of IL-1B (−511) or allele 1 of IL-1RN (+2018), oran allele that is in linkage disequilibrium with one of theaforementioned alleles. In a preferred embodiment, this allelic patternpermits the diagnosis of a restenosis disorder.

In another embodiment, the method of the invention may be employed bydetecting the presence of an IL-1 associated polymorphism that is inlinkage disequilibrium with one or more of the aforementionedrestenosis-predictive alleles. For example, the following alleles of theIL-1 (44112332) haplotype are known to be in linkage disequilibrium:

allele 4 of the 222/223 marker of IL-1A allele 4 of the gz5/gz6 markerof IL-1A allele 1 of the −889 marker of IL-1A allele 1 of the +3954marker of IL-1B allele 2 of the −511 marker of IL-1B allele 3 of thegaat.p33330 marker allele 3 of the Y31 marker allele 2 of the VNTR or(+2018) marker of IL-1RN

Also, the following alleles of the IL-1 (33221461) haplotype are inlinkage disequilibrium:

allele 3 of the 222/223 marker of IL-1A allele 3 of the gz5/gz6 markerof IL-1A allele 2 of the −889 marker of IL-1A allele 2 of the +3954marker of IL-1B allele 1 of the −511 marker of IL-1B allele 4 of thegaat.p33330 marker allele 6 of the Y31 marker allele 1 of the VNTR or(+2018) marker of IL-1RN

A restenosis associated allele can be detected by any of a variety ofavailable techniques, including: 1) performing a hybridization reactionbetween a nucleic acid sample and a probe that is capable of hybridizingto the allele; 2) sequencing at least a portion of the allele; or 3)determining the electrophoretic mobility of the allele or fragmentsthereof (e.g., fragments generated by endonuclease digestion). Theallele can optionally be subjected to an amplification step prior toperformance of the detection step. Preferred amplification methods areselected from the group consisting of: the polymerase chain reaction(PCR), the ligase chain reaction (LCR), strand displacementamplification (SDA), cloning, and variations of the above (e.g. RT-PCRand allele specific amplification). Oligonucleotides necessary foramplification may be selected for example, from within the IL-1 geneloci, either flanking the marker of interest (as required for PCRamplification) or directly overlapping the marker (as in ASOhybridization). In a particularly preferred embodiment, the sample ishybridized with a set of primers, which hybridize 5′ and 3′ in a senseor antisense sequence to the restenosis associated allele, and issubjected to a PCR amplification.

A restenosis associated allele may also be detected indirectly, e.g. byanalyzing the protein product encoded by the DNA. For example, where themarker in question results in the translation of a mutant protein, theprotein can be detected by any of a variety of protein detectionmethods. Such methods include immunodetection and biochemical tests,such as size fractionation, where the protein has a change in apparentmolecular weight either through truncation, elongation, altered foldingor altered post-translational modifications.

In another aspect, the invention features kits for performing theabove-described assays. The kit can include a nucleic acid samplecollection means and a means for determining whether a subject carries arestenosis associated allele. The kit may also contain a control sampleeither positive or negative or a standard and/or an algorithmic devicefor assessing the results and additional reagents and componentsincluding: DNA amplification reagents, DNA polymerase, nucleic acidamplification reagents, restrictive enzymes, buffers, a nucleic acidsampling device, DNA purification device, deoxynucleotides,oligonucleotides (e.g. probes and primers) etc.

As described above, the control samples may be positive or negativecontrols. Further, the control sample may contain the positive (ornegative) products of the allele detection technique employed. Forexample, where the allele detection technique is PCR amplification,followed by size fractionation, the control sample may comprise DNAfragments of the appropriate size. Likewise, where the allele detectiontechnique involves detection of a mutated protein, the control samplemay comprise a sample of mutated protein. However, it is preferred thatthe control sample comprises the material to be tested. For example, thecontrols may be a sample of genomic DNA or a cloned portion of the IL-1gene cluster. Preferably, however, the control sample is a highlypurified sample of genomic DNA where the sample to be tested is genomicDNA.

The oligonucleotides present in said kit may be used for PCRamplification of the region of interest or for direct allele specificoligonucleotide (ASO) hybridization to the markers in question. Thus,the oligonucleotides may either flank the marker of interest (asrequired for PCR amplification) or directly overlap the marker (as inASO hybridization).

Such oligonucleotides can include, but are not limited to:

(SEQ ID No. 1) 5′ ATG GTT TTA GAA ATC ATC AAG CCT AGG GCA 3′ and (SEQ IDNo. 2) 5′ AAT GAA AGG AGG GGA GGA TGA CAG AAA TGT 3′

which can be used to amplify the human IL-1A (+4845) polymorphic locus;

5′ TGG CAT TGA TCT GGT TCA TC 3′ (SEQ ID No. 3) and 5′ GTT TAG GAA TCTTCC CAC TT-3′ (SEQ ID No. 4)

which can be used to amplify the human IL-1B (−511) polymorphic locus;

(SEQ ID No. 5) 5′-CTC AGG TGT CCT CGA AGA AAT CAA A-3′ and (SEQ ID No.6) 5′ GCT TTT TTG CTG TGA GTC CCG-3′

which can be used to amplify the human IL-1B (+3954) polymorphic locus;

5′-CTC.AGC.AAC.ACT.CCT.AT-3′ (SEQ ID NO. 7) and5′-TCC.TGG.TCT.GCA.GCT.AA-3′ (SEQ ID NO. 8)

which can be used to amplify the human IL-1RN (VNTR) polymorphic locus;

(SEQ ID NO. 9) 5′-CTA TCT GAG GAA CAA CCA ACT AGT AGC-3′ and (SEQ ID NO.10) 5′-TAG GAC ATT GCA CCT AGG GTT TGT -3′

which can be used to amplify the human IL-1 RN (+2018) polymorphiclocus;

(SEQ. ID No. 11) 5′ ATT TTT TTA TAA ATC ATC AAG CCT AGG GCA 3′ and (SEQ.ID No. 12) 5′ AAT TAA AGG AGG GAA GAA TGA CAG AAA TGT 3′

which can also be used to amplify the human IL-1A (+4845) polymorphiclocus;

(SEQ. ID NO. 13) 5′-AAG CTT GTT CTA CCA CCT GAA CTA GGC.-3′ and (SEQ. IDNO. 14) 5′-TTA CAT ATG AGC CTT CCA TG.-3′

which can be used to amplify the human IL-1A (−889) polymorphic locus;

Information obtained using the assays and kits described herein (aloneor in conjunction with information on another genetic defect orenvironmental factor, which contributes to restenosis) is useful fordetermining whether a non-symptomatic subject has or is likely todevelop restenosis. In addition, the information can allow a morecustomized approach to preventing the onset or progression ofrestenosis. For example, this information can enable a clinician to moreeffectively prescribe a therapy that will address the molecular basis ofrestenosis. In yet a further aspect, the invention features methods fortreating or preventing the development of restenosis in a subject byadministering to the subject an appropriate restenosis therapeutic ofthe invention. In still another aspect, the invention provides in vitroor in vivo assays for screening test compounds to identify restenosistherapeutics. In one embodiment, the assay comprises contacting a celltransfected with a restenosis causative mutation that is operably linkedto an appropriate promoter with a test compound and determining thelevel of expression of a protein in the cell in the presence and in theabsence of the test compound. In a preferred embodiment, the restenosiscausative mutation results in decreased production of IL-1 receptorantagonist, and increased production of the IL-1 receptor antagonist inthe presence of the test compound indicates that the compound is anagonist of IL-1 receptor antagonist activity. In another preferredembodiment, the restenosis causative mutation results in increasedproduction of IL-1α or IL-1β, and decreased production of IL-1α or IL-1βin the presence of the test compound indicates that the compound is anantagonist of IL-1α or IL-1β activity. In another embodiment, theinvention features transgenic non-human animals and their use inidentifying antagonists of IL-1α or IL-1β activity or agonists of IL-1Raactivity.

Other embodiments and advantages of the invention are set forth in partin the description which follows, and will be obvious from thisdescription.

3. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the nucleic acid sequence for IL-1A (GEN X03833; SEQ ID No.15).

FIG. 2 shows the nucleic acid sequence for IL-1B (GEN X04500; SEQ ID No.16).

FIG. 3 shows the nucleic acid sequence for the secreted IL-1RN (GENX64532; SEQ ID No. 17).

FIG. 4 depicts the organization of the IL-1 genes, and associatedpolymorphic loci, on human chromosome 2.

FIG. 5 shows linkage disequilibrium values for the IL-1 polymorphic lociin a Caucasian population.

FIG. 6 is a bar graph illustrating the frequency of particular IL-1polymorphic allelic patterns in a Caucasian population.

FIG. 7 indicates the relative risk for restenosis associated with eachof the IL-1 polymorphic patterns.

FIG. 8 indicates the association between homozygous and heterozygousallelic patterns at the IL-1RN (+2018) locus and the occurrence ofrestenosis and target vessel revascularization.

FIG. 9 is a graph showing the odds ratios for clinical events andangiographic restenosis associated with the presence of the IL-1RN*2allele for the whole population (left panel) and patients <60 years(right panel)

FIG. 10. is a bar graph showing the decrease in the incidence ofrestenosis and target vessel revascularization (TVR) in patients <60years with the increase in the number of IL-1RN*2 alleles.

4. DETAILED DESCRIPTION OF THE INVENTION 4.1 Definitions

For convenience, the meaning of certain terms and phrases employed inthe specification, examples, and appended claims are provided below.

The term “allele” refers to the different sequence variants found atdifferent polymorphic regions. For example, IL-1RN (VNTR) has at leastfive different alleles. The sequence variants may be single or multiplebase changes, including without limitation insertions, deletions, orsubstitutions, or may be a variable number of sequence repeats.

The term “allelic pattern” refers to the identity of an allele oralleles at one or more polymorphic regions. For example, an allelicpattern may consist of a single allele at a polymorphic site, as forIL-1RN (VNTR) allele 1, which is an allelic pattern having at least onecopy of IL-1RN allele 1 at the VNTR of the IL-1RN gene loci.Alternatively, an allelic pattern may consist of either a homozygous orheterozygous state at a single polymorphic site. For example, IL1-RN(VNTR) allele 2,2 is an allelic pattern in which there are two copies ofthe second allele at the VNTR marker of IL-1RN and that corresponds tothe homozygous IL-RN (VNTR) allele 2 state. Alternatively, an allelicpattern may consist of the identity of alleles at more than onepolymorphic site.

The term “antibody” as used herein is intended to refer to a bindingagent including a whole antibody or a binding fragment thereof which isspecifically reactive with an IL-1B polypeptide. Antibodies can befragmented using conventional techniques and the fragments screened forutility in the same manner as described above for whole antibodies. Forexample, F(ab)₂ fragments can be generated by treating an antibody withpepsin. The resulting F(ab)₂ fragment can be treated to reduce disulfidebridges to produce Fab fragments. The antibody of the present inventionis further intended to include bispecific, single-chain, and chimericand humanized molecules having affinity for an IL-1B polypeptideconferred by at least one CDR region of the antibody.

“Biological activity” or “bioactivity” or “activity” or “biologicalfunction”, which are used interchangeably, for the purposes herein meansan effector or antigenic function that is directly or indirectlyperformed by an IL-1 polypeptide (whether in its native or denaturedconformation), or by any subsequence thereof. Biological activitiesinclude binding to a target peptide, e.g., an IL-1 receptor. An IL-1bioactivity can be modulated by directly affecting an IL-1 polypeptide.Alternatively, an IL-1 bioactivity can be modulated by modulating thelevel of an IL-1 polypeptide, such as by modulating expression of anIL-1 gene.

As used herein the term “bioactive fragment of an IL-1 polypeptide”refers to a fragment of a full-length IL-1 polypeptide, wherein thefragment specifically mimics or antagonizes the activity of a wild-typeIL-1 polypeptide. The bioactive fragment preferably is a fragmentcapable of interacting with an interleukin receptor.

The term “an aberrant activity”, as applied to an activity of apolypeptide such as IL-1, refers to an activity which differs from theactivity of the wild-type or native polypeptide or which differs fromthe activity of the polypeptide in a healthy subject. An activity of apolypeptide can be aberrant because it is stronger than the activity ofits native counterpart. Alternatively, an activity can be aberrantbecause it is weaker or absent relative to the activity of its nativecounterpart. An aberrant activity can also be a change in an activity.For example an aberrant polypeptide can interact with a different targetpeptide. A cell can have an aberrant IL-1 activity due to overexpressionor underexpression of an IL-1 locus gene encoding an IL-1 locuspolypeptide.

“Cells”, “host cells” or “recombinant host cells” are terms usedinterchangeably herein to refer not only to the particular subject cell,but to the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact be identicalto the parent cell, but is still included within the scope of the termas used herein.

A “chimera,” “mosaic,” “chimeric mammal” and the like, refers to atransgenic mammal with a knock-out or knock-in construct in at leastsome of its genome-containing cells.

The terms “control” or “control sample” refer to any sample appropriateto the detection technique employed. The control sample may contain theproducts of the allele detection technique employed or the material tobe tested. Further, the controls may be positive or negative controls.By way of example, where the allele detection technique is PCRamplification, followed by size fractionation, the control sample maycomprise DNA fragments of an appropriate size. Likewise, where theallele detection technique involves detection of a mutated protein, thecontrol sample may comprise a sample of a mutant protein. However, it ispreferred that the control sample comprises the material to be tested.For example, the controls may be a sample of genomic DNA or a clonedportion of the IL-1 gene cluster. However, where the sample to be testedis genomic DNA, the control sample is preferably a highly purifiedsample of genomic DNA.

A “cardiovascular disease” is a cardiovascular disorder, as definedherein, characterized by clinical events including clinical symptoms andclinical signs. Clinical symptoms are those experiences reported by apatient that indicate to the clinician the presence of pathology.Clinical signs are those objective findings on physical or laboratoryexamination that indicate to the clinician the presence of pathology.“Cardiovascular disease” includes both “coronary artery disease” and“peripheral vascular disease,” both terms being defined below. Clinicalsymptoms in cardiovascular disease include chest pain, shortness ofbreath, weakness, fainting spells, alterations in consciousness,extremity pain, paroxysmal nocturnal dyspnea, transient ischemic attacksand other such phenomena experienced by the patient. Clinical signs incardiovascular disease include such findings as EKG abnormalities,altered peripheral pulses, arterial bruits, abnormal heart sounds, ralesand wheezes, jugular venous distention, neurological alterations andother such findings discerned by the clinician. Clinical symptoms andclinical signs can combine in a cardiovascular disease such as amyocardial infarction (MI) or a stroke (also termed a “cerebrovascularaccident” or “CVA”), where the patient will report certain phenomena(symptoms) and the clinician will perceive other phenomena (signs) allindicative of an underlying pathology. “Cardiovascular disease” includesthose diseases related to the cardiovascular disorders of fragile plaquedisorder, occlusive disorder and stenosis. For example, a cardiovasculardisease resulting from a fragile plaque disorder, as that term isdefined below, can be termed a “fragile plaque disease.” Clinical eventsassociated with fragile plaque disease include those signs and symptomswhere the rupture of a fragile plaque with subsequent acute thrombosisor with distal embolization are hallmarks. Examples of fragile plaquedisease include certain strokes and myocardial infarctions. As anotherexample, a cardiovascular disease resulting from an occlusive disordercan be termed an “occlusive disease.” Clinical events associated withocclusive disease include those signs and symptoms where the progressiveocclusion of an artery affects the amount of circulation that reaches atarget tissue. Progressive arterial occlusion may result in progressiveischemia that may ultimately progress to tissue death if the amount ofcirculation is insufficient to maintain the tissues. Signs and symptomsof occlusive disease include claudication, rest pain, angina, andgangrene, as well as physical and laboratory findings indicative ofvessel stenosis and decreased distal perfusion. As yet another example,a cardiovascular disease resulting from restenosis can be termed anin-stent stenosis disease. In-stent stenosis disease includes the signsand symptoms resulting from the progressive blockage of an arterialstent that has been positioned as part of a procedure like apercutaneous transluminal angioplasty, where the presence of the stentis intended to help hold the vessel in its newly expanded configuration.The clinical events that accompany in-stent stenosis disease are thoseattributable to the restenosis of the reconstructed artery.

A “cardiovascular disorder” refers broadly to both to coronary arterydisorders and peripheral arterial disorders. The term “cardiovasculardisorder” can apply to any abnormality of an artery, whether structural,histological, biochemical or any other abnormality. This term includesthose disorders characterized by fragile plaque (termed herein “fragileplaque disorders”), those disorders characterized by vaso-occlusion(termed herein “occlusive disorders”), and those disorders characterizedby restenosis. A “cardiovascular disorder” can occur in an arteryprimarily, that is, prior to any medical or surgical intervention.Primary cardiovascular disorders include, among others, atherosclerosis,arterial occlusion, aneurysm formation and thrombosis. A “cardiovasculardisorder” can occur in an artery secondarily, that is, following amedical or surgical intervention. Secondary cardiovascular disordersinclude, among others, post-traumatic aneurysm formation, restenosis,and post-operative graft occlusion.

A “cardiovascular disorder causative functional mutation” refers to amutation which causes or contributes to the development of acardiovascular disorder in a subject. Preferred mutations occur withinthe IL-1 complex. A cardiovascular disorder causative functionalmutation occurring within an IL-1 gene (e.g. IL-1A, IL-1B or IL-1RN) ora gene locus, which is linked thereto, may alter, for example, the openreading frame or splicing pattern of the gene, thereby resulting in theformation of an inactive or hypoactive gene product. For example, amutation which occurs in intron 6 of the IL-1A locus corresponds to avariable number of tandem repeat 46 bp sequences corresponding to fromfive to 18 repeat units (Bailly, et al. (1993) Eur. J. Immunol. 23:1240-45). These repeat sequences contain three potential binding sitesfor transcriptional factors: an SP1 site, a viral enhancer element, anda glucocorticoid-responsive element; therefore individuals carryingIL-1A intron 6 VNTR alleles with large numbers of repeat units may besubject to altered transcriptional regulation of the IL-1A gene andconsequent perturbations of inflammatory cytokine production. Indeed,there is evidence that increased repeat number at this polymorphic IL-1Alocus leads to decreased IL-1α synthesis (Bailly et al. (1996) MolImmunol 33: 999-1006). Alternatively, a mutation can result in ahyperactive gene product. For example, allele 2 of the IL-1B (G at+6912) polymorphism occurs in the 3′ UTR (untranslated region) of theIL-1B mRNA and is associated with an approximately four-fold increase inthe steady state levels of both IL-1B mRNA and IL-1B protein compared tothose levels associated with allele 1 of the IL-1B gene at +6912).Further, an IL-1B (−511) mutation occurs near a promoter binding sitefor a negative glucocorticoid response element (Zhang et al. (1997) DNACell Biol 16: 145-52). This element potentiates a four-fold repressionof IL-1B expression by dexamethosone and a deletion of this negativeresponse elements causes a 2.5-fold increase in IL-1B promoter activity.The IL-1B (−511) polymorphism may thus directly affect cytokineproduction and inflammatory responses. These examples demonstrate thatgenetic variants occurring in the IL-1A or IL-1B gene can directly leadto the altered production or regulation of IL-1 cytokine activity.

A “cardiovascular disorder therapeutic” refers to any agent ortherapeutic regimen (including pharmaceuticals, nutraceuticals andsurgical means) that prevents or postpones the development of or reducesthe extent of an abnormality constitutive of a cardiovascular disorderin a subject. Cardiovascular disorder therapeutics can be directed tothe treatment of any cardiovascular disorder, including fragile plaquedisorder, occlusive disorder and restenosis. Examples of therapeuticagents directed to each category of cardiovascular disorder are providedherein. It is understood that a therapeutic agent may be useful for morethan one category of cardiovascular disorder. The therapeutic can be apolypeptide, peptidomimetic, nucleic acid or other inorganic or organicmolecule, preferably a “small molecule” including vitamins, minerals andother nutrients. Preferably the therapeutic can modulate at least oneactivity of an IL-1 polypeptide, e.g., interaction with a receptor, bymimicking or potentiating (agonizing) or inhibiting (antagonizing) theeffects of a naturally-occurring polypeptide. An IL-1 agonist can be awild-type protein or derivative thereof having at least one bioactivityof the wild-type, e.g., receptor binding activity. An IL-1 agonist canalso be a compound that upregulates expression of a gene or whichincreases at least one bioactivity of a protein. An IL-1 agonist canalso be a compound which increases the interaction of a polypeptide withanother molecule, e.g., a receptor. An IL-1 antagonist can be a compoundwhich inhibits or decreases the interaction between a protein andanother molecule, e.g., a receptor or an agent that blocks signaltransduction or post-translation processing (e.g., IL-1 convertingenzyme (ICE) inhibitor). Accordingly, a preferred antagonist is acompound which inhibits or decreases binding to a receptor and therebyblocks subsequent activation of the receptor. An IL-1 antagonist canalso be a compound that downregulates expression of a gene or whichreduces the amount of a protein present. The antagonist can be adominant negative form of a polypeptide, e.g., a form of a polypeptidewhich is capable of interacting with a target peptide, e.g., a receptor,but which does not promote the activation of the receptor. Theantagonist can also be a nucleic acid encoding a dominant negative formof a polypeptide, an antisense nucleic acid, or a ribozyme capable ofinteracting specifically with an RNA. Yet other antagonists aremolecules which bind to a polypeptide and inhibit its action. Suchmolecules include peptides, e.g., forms of target peptides which do nothave biological activity, and which inhibit binding to receptors. Thus,such peptides will bind to the active site of a protein and prevent itfrom interacting with target peptides. Yet other antagonists includeantibodies that specifically interact with an epitope of a molecule,such that binding interferes with the biological function of thepolypeptide. In yet another preferred embodiment, the antagonist is asmall molecule, such as a molecule capable of inhibiting the interactionbetween a polypeptide and a target receptor. Alternatively, the smallmolecule can function as an antagonist by interacting with sites otherthan the receptor binding site. Preferred therapeutics include lipidlowering drugs, antiplatelet agents, anti-inflammatory agents andantihypertensive agents.

“Cerebrovascular disease,” as used herein, is a type of peripheralvascular disease (as defined below) where the peripheral vessel blockedis part of the cerebral circulation. The cerebral circulation includesthe carotid and the vertebral arterial systems. This definition ofcerebrovascular disease is intended specifically to include intracranialhemorrhage that does not occur as a manifestation of an arterialblockage. Blockage can occur suddenly, by mechanisms such as plaquerupture or embolization. Blockage can occur progressively, withnarrowing of the artery via myointimal hyperplasia and plaque formation.Blockage can be complete or partial. Certain degrees and durations ofblockage result in cerebral ischemia, a reduction of blood flow thatlasts for several seconds to minutes. The prolongation of cerebralischemia can result in cerebral infarction. Ischemia and infarction canbe focal or widespread. Cerebral ischemia or infarction can result inthe abrupt onset of a non-convulsive focal neurological defect, aclinical event termed a “stroke” or a “cerebrovascular accident (CVA)”.Cerebrovascular disease has two broad categories of pathologies:thrombosis and embolism. Thrombotic strokes occur without warningsymptoms in 80-90% of patients; between 10 and 20% of thrombotic strokesare heralded by transient ischemic attacks. A cerebrovascular diseasecan be associated with a fragile plaque disorder. The signs and symptomsof this type of cerebrovascular disease are those associated withfragile plaque, including stroke due to sudden arterial blockage withthrombus or embolus formation. A cerebrovascular disease can beassociated with occlusive disorder. The signs and symptoms of this typeof cerebrovascular disease relate to progressive blockage of blood flowwith global or local cerebral ischemia. In this setting, neurologicalchanges can be seen, including stroke.

A “clinical event” is an occurrence of clinically discernible signs of adisease or of clinically reportable symptoms of a disease. “Clinicallydiscernible” indicates that the sign can be appreciated by a health careprovider. “Clinically reportable” indicates that the symptom is the typeof phenomenon that can be described to a health care provider. Aclinical event may comprise clinically reportable symptoms even if theparticular patient cannot himself or herself report them, as long asthese are the types of phenomena that are generally capable ofdescription by a patient to a health care provider.

A “coronary artery disease” (“CAD”) refers to a vascular disorderrelating to the blockage of arteries serving the heart. Blockage canoccur suddenly, by mechanisms such as plaque rupture or embolization.Blockage can occur progressively, with narrowing of the artery viamyointimal hyperplasia and plaque formation. Those clinical signs andsymptoms resulting from the blockage of arteries serving the heart aremanifestations of coronary artery disease. Manifestations of coronaryartery disease include angina, ischemia, myocardial infarction,cardiomyopathy, congestive heart failure, arrhythmias and aneurysmformation. It is understood that fragile plaque disease in the coronarycirculation is associated with arterial thrombosis or distalembolization that manifests itself as a myocardial infarction. It isunderstood that occlusive disease in the coronary circulation isassociated with arterial stenosis accompanied by anginal symptoms, acondition commonly treated with pharmacological interventions and withangioplasty.

A “disease” is a disorder characterized by clinical events includingclinical signs and clinical symptoms. The diseases discussed hereininclude cardiovascular disease, peripheral vascular disease, CAD,cerebrovascular disease, and those diseases in any anatomic locationassociated with fragile plaque disorder, with occlusive disorder or withrestenosis.

A “disorder associated allele” or “an allele associated with a disorder”refers to an allele whose presence in a subject indicates that thesubject has or is susceptible to developing a particular disorder. Onetype of disorder associated allele is a “cardiovascular disorderassociated allele,” the presence of which in a subject indicates thatthe subject has or is susceptible to developing a cardiovasculardisorder. These include broadly within their scope alleles which areassociated with “fragile plaque disorders,” alleles associated with“occlusive disorders,” and alleles associated with restenosis. Examplesof alleles associated with “fragile plaque disorders” include thosealleles comprising the IL-1 pattern 1—i.e. allele 2 of the IL-1A +4825;allele 2 of the +3954 marker of IL-1B; and allele 1 of the +2018 markerof IL-1RN; and allele 1 of the (−511) marker of the IL-1B gene or anallele that is in linkage disequilibrium with one of the aforementionedalleles. Examples of alleles associated with “occlusive disorders”include those comprising the IL-1 pattern 2—i.e. allele 1 of the IL-1A+4825; allele 1 of the +3954 marker of IL-1B; and allele 2 of the +2018marker of IL-1RN; and allele 2 of the (−511) marker of the IL-1B gene oran allele that is in linkage disequilibrium with one of theaforementioned alleles. Examples of alleles associated with restenosisinclude the combination of either allele 1 of the +4825 marker of IL-1Aor allele 1 of the +3954 marker as combined with either allele 1 of the−511 marker of IL-1B or allele 1 of the +2018 marker of IL-1RN, or anallele that is in linkage disequilibrium with one of the aforementionedalleles. A “periodontal disorder associated allele” refers to an allelewhose presence in a subject indicates that the subject has or issusceptible to developing a periodontal disorders.

The phrases “disruption of the gene” and “targeted disruption” or anysimilar phrase refers to the site specific interruption of a native DNAsequence so as to prevent expression of that gene in the cell ascompared to the wild-type copy of the gene. The interruption may becaused by deletions, insertions or modifications to the gene, or anycombination thereof.

The term “haplotype” as used herein is intended to refer to a set ofalleles that are inherited together as a group (are in linkagedisequilibrium) at statistically significant levels (p_(corr)<0.05). Asused herein, the phrase “an IL-1 haplotype” refers to a haplotype in theIL-1 loci.

The term “hyperplasia” as used herein is intended to refer to anabnormal or unusual increase in growth or division of the cellscomposing a tissue or organ. It is understood that the term“hyperplasia,” as used herein, encompasses a wide variety of specificproliferative states including “neointimal hyperplasia” or “neointimalgrowth,” which refers to hyperplasia of the of cells in the endotheliallayer of a blood vessel and “myointimal hyperplasia” or “myointimalgrowth,” which refers to an abnormal proliferation of smooth musclecells of the vascular wall. The terms myointimal and neointimal are usedinterchangeably herein.

An “IL-1 agonist” as used herein refers to an agent that mimics,upregulates (potentiates or supplements) or otherwise increases an IL-1bioactivity or a bioactivity of a gene in an IL-1 biological pathway.IL-1 agonists may act on any of a variety of different levels, includingregulation of IL-1 gene expression at the promoter region, regulation ofmRNA splicing mechanisms, stabilization of mRNA, phosphorylation ofproteins for translation, conversion of proIL-1 to mature IL-1 andsecretion of IL-1. Agonists that increase IL-1 synthesis include:lipopolysaccharides, IL-1B, cAMP inducing agents, NfκB activatingagents, AP-1 activating agents, TNF-α, oxidized LDL, advancedglycosylation end products (AGE), sheer stress, hypoxia, hyperoxia,ischemia reperfusion injury, histamine, prostaglandin E 2 (PGE2), IL-2,IL-3, IL-12, granulocyte macrophage-colony stimulating factor (GM-CSF),monocyte colony stimulating factor (M-CSF), stem cell factor, plateletderived growth factor (PDGF), complement C5A, complement C5b9, fibrindegradation products, plasmin, thrombin, 9-hydroxyoctadecaenoic acid,13-hydroxyoctadecaenoic acid, platelet activating factor (PAF), factorH, retinoic acid, uric acid, calcium pyrophosphate, polynucleosides,c-reactive protein, α-antitrypsin, tobacco antigen, collagen, β-1integrins, LFA-3, anti-HLA-DR, anti-IgM, anti-CD3, phytohemagglutinin(CD2), sCD23, ultraviolet B radiation, gamma radiation, substance P,isoproterenol, methamphetamine and melatonin. Agonists that stabilizeIL-1 mRNA include bacterial endotoxin and IL-1. Other agonists, thatfunction by increasing the number of IL-1 type 1 receptors available,include IL-1, PKC activators, dexamethasone, IL-2, IL-4 and PGE2. Otherpreferred antagonists interfere or inhibit signal transduction factorsactivated by IL-1 or utilized in an IL-1 signal transduction pathway(e.g NFκB and AP-1, P13 kinase, phospholipase A2, protein kinase C,JNK-1, 5-lipoxygenase, cyclooxygenase 2, tyrosine phosphorylation, INOSpathway, Rac, Ras, TRAF). Still other agonists increase the bioactivityof genes whose expression is induced by IL-1, including: IL-1, IL-1Ra,TNF, IL-2, IL-3, IL-6, IL-12, GM-CSF, G-CSF, TGF-β, fibrinogen,urokinase plasminogen inhibitor, Type I and type 2 plasminogen activatorinhibitor, p-selectin (CD62), fibrinogen receptor, CD-II/CD18, proteasenexin-1, CD44, Matrix metalloproteinase-1 (MMP-1), MMP-3, Elastase,Collagenases, Tissue inhibitor of metalloproteinases-1 (TIMP-1),Collagen, Triglyceride increasing Apo CIII, Apolipoprotein, ICAM-1,ELAM-1, VCAM-1, L-selectin, Decorin, stem cell factor, Leukemiainhibiting factor, IFNα,β,γ, L-8, IL-2 receptor, IL-3 receptor, IL-5receptor, c-kit receptor, GM-CSF receptor, Cyclooxygenase-2 (COX-2),Type 2 phospholipase A2, Inducible nitric oxide synthase (iNOS),Endothelin-1,3, Gamma glutamyl transferase, Mn superoxide dismutase, C—reactive protein, Fibrinogen, Serum amyloid A, Metallothioneins,Ceruloplasmin, Lysozyme, Xanthine dehydrogenase, Xanthine oxidase,Platelet derived growth factor A chain (PDGF), Melanoma growthstimulatory activity (gro-α,β,γ), Insulin-like growth factor-1 (IGF-1),Activin A, Pro-opiomelanocortiotropin, corticotropin releasing factor, Bamyloid precursor, Basement membrane protein-40, Laminin B1 and B2,Constitutive heat shock protein p70, P42 mitogen, activating proteinkinase, ornithine decarboxylase, heme oxygenase and G-protein αsubunit).

An “IL-1 antagonist” as used herein refers to an agent thatdownregulates or otherwise decreases an IL-1 bioactivity. IL-1antagonists may act on any of a variety of different levels, includingregulation of IL-1 gene expression at the promoter region, regulation ofmRNA splicing mechanisms, stabilization of mRNA, phosphorylation ofproteins for translation, conversion of proIL-1 to mature IL-1 andsecretion of IL-1. Antagonists of IL-1 production include:corticosteroids, lipoxygenase inhibitors, cyclooxygenase inhibitors,γ-interferon, IL-4, IL-10, IL-13, transforming growth factor 0 (TGF-β,ACE inhibitors, n-3 polyunsaturated fatty acids, antioxidants and lipidreducing agents. Antagonists that destabilize IL-1mRNA include agentsthat promote deadenylation. Antagonists that inhibit or preventphosphorylation of IL-1 proteins for translation includepyridinyl-imadazole compounds, such as tebufelone and compounds thatinhibit microtubule formation (e.g. colchicine, vinblastine andvincristine). Antagonists that inhibit or prevent the conversion ofproIL-1 to mature IL-1 include interleukin converting enzyme (ICE)inhibitors, such as εICE isoforms, ICE α, γ, and γ isoform antibodies,CXrm-A, transcript X, endogenous tetrapeptide competitive substrateinhibitor, trypsin, elastase, chyrnotrypsin, chymase, and othernonspecific proteases. Antagonists that prevent or inhibit the scretionof IL-1 include agents that block anion transport. Antagonists thatinterfere with IL-1 receptor interactions, include: agents that inhibitglycosylation of the type I IL-1 receptor, antisense oligonucleotidesagainst IL-1RI, antibodies to IL-1RI and antisense oligonucleotidesagainst IL-1RacP. Other antagonists, that function by decreasing thenumber of IL-1 type 1 receptors available, include TGF-β, COXinhibitors, factors that increase IL-1 type II receptors, dexamethasone,PGE2, IL-1 and IL-4. Other preferred antagonists interfere or inhibitsignal transduction factors activated by IL-1 or utilized in an IL-1signal transduction pathway (e.g NFκB and AP-1, PI3 kinase,phospholipase A2, protein kinase C, JNK-1,5-lipoxygenase, cyclooxygenase2, tyrosine phosphorylation, iNOS pathway, Rac, Ras, TRAF). Still otherantagonists interfere with the bioactivity of genes whose expression isinduced by IL-1, including: IL-1, IL-1Ra, TNF, IL-2, IL-3, IL-6, IL-12,GM-CSF, G-CSF, TGF-β, fibrinogen, urokinase plasminogen inhibitor, Type1 and type 2 plasminogen activator inhibitor, p-selectin (CD62),fibrinogen receptor, CD-11/CD18, protease nexin-1, CD44, Matrixmetalloproteinase-1 (MMP-1), MMP-3, Elastase, Collagenases, Tissueinhibitor of metalloproteinases-1 (TIMP-1), Collagen, Triglycerideincreasing Apo CIII, Apolipoprotein, ICAM-1, ELAM-1, VCAM-1, L-selectin,Decorin, stem cell factor, Leukemia inhibiting factor, IFNα,β,γ, L-8,IL-2 receptor, IL-3 receptor, IL-5 receptor, c-kit receptor, GM-CSFreceptor, Cyclooxygenase-2 (COX-2), Type 2 phospholipase A2, Induciblenitric oxide synthase (iNOS), Endothelin-1,3, Gamma glutamyltransferase, Mn superoxide dismutase, C— reactive protein, Fibrinogen,Serum amyloid A, Metallothioneins, Ceruloplasmin, Lysozyme, Xanthinedehydrogenase, Xanthine oxidase, Platelet derived growth factor A chain(PDGF), Melanoma growth stimulatory activity (gro-α,β,γ), Insulin-likegrowth factor-1 (IGF-1), Activin A, Pro-opiomelanocortiotropin,corticotropin releasing factor, B amyloid precursor, Basement membraneprotein-40, Laminin B1 and B2, Constitutive heat shock protein p70, P42mitogen, activating protein kinase, ornithine decarboxylase, hemeoxygenase and G-protein α subunit). Other preferred antagonists include:hymenialdisine, herbimycines (e.g. herbamycin A), CK-103A and itsderivatives (e.g. 4,6-dihydropyridazino[4,5-c]pyridazin-5 (1H)-one),CK-119, CK-122, iodomethacin, aflatoxin B1, leptin, heparin, bicyclicimidazoles (e.g SB203580), PD15306HCl, podocarpic acid derivatives,M-20, Human [Gly2] Glucagon-like peptide-2, FR167653, Steroidderivatives, glucocorticoids, Quercetin, Theophylline, NO-synthetaseinhibitors, RWJ 68354, Euclyptol (1.8-cineole), Magnosalin,N-Acetylcysteine, Alpha-Melatonin-Stimulating Hormone (α-MSH), Triclosan(2,4,4′-trichloro-2′-hydroxyldiphenyl ether), Prostaglandin E2 and4-aminopyridine Ethacrynic acid and4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), Glucose,Lipophosphoglycan, aspirin, Catabolism-blocking agents, Diacerhein,Thiol-modulating agents, Zinc, Morphine, Leukotriene biosynthesisinhibitors (e.g. MK886), Platelet-activating factor receptor antagonists(e.g. WEB 2086), Amiodarone, Tranilast, S-methyl-L-thiocitrulline,Beta-adrenoreceptor agonists (e.g. Procaterol, Clenbuterol, Fenoterol,Terbutaline, Hyaluronic acid, anti-TNF-α antibodies, anti-IL-1αautoantibodies, IL-1 receptor antagonist, IL-1R-associated kinase,soluble TNF receptors and antiinflammatory cytokines (e.g IL-4, IL-13,IL-10, IL-6, TGF-β, angiotensin II, Soluble IL-1 type II receptor,Soluble IL-1 type I receptor, Tissue plasminogen activator, Zinc fingerprotein A20 IL-1 Peptides (e.g (Thr-Lys-Pro-Arg) (Tuftsin),(Ile-Thr-Gly-Ser-Glu) IL-1-alpha, Val-Thr-Lys-Phe-Tyr-Phe,Val-Thr-Asp-Phe-Tyr-Phe, Interferon alpha2b, Interferon beta, IL-1-betaanalogues (e.g. IL-1-beta tripeptide: Lys-D-Pro-Thr), glycosylatedIL-1-alpha, and IL-1ra peptides.

The terms “IL-1 gene cluster” and “IL-1 loci” as used herein include allthe nucleic acid at or near the 2q13 region of chromosome 2, includingat least the IL-1A, IL-1B and IL-1RN genes and any other linkedsequences. (Nicklin et al., Genomics 19: 382-84, 1994). The terms“IL-1A”, “IL-1B”, and “IL-1RN” as used herein refer to the genes codingfor IL-1, IL-1, and IL-1 receptor antagonist, respectively. The geneaccession number for IL-1A, IL-1B, and IL-1RN are X03833, X04500, andX64532, respectively.

“IL-1 functional mutation” refers to a mutation within the IL-1 genecluster that results in an altered phenotype (i.e. effects the functionof an IL-1 gene or protein). Examples include: IL-1A(+4845) allele 2,IL-1B (+3954) allele 2, IL-1B (+6912) allele 2 and IL-1RN (+2018) allele2.

“IL-1X (Z) allele Y” refers to a particular allelic form, designated Y,occurring at an IL-1 locus polymorphic site in gene X, wherein X isIL-1A, B, or RN or some other gene in the IL-1 gene loci, and positionedat or near nucleotide Z, wherein nucleotide Z is numbered relative tothe major transcriptional start site, which is nucleotide +1, of theparticular IL-1 gene X. As further used herein, the term “IL-1X allele(Z)” refers to all alleles of an IL-1 polymorphic site in gene Xpositioned at or near nucleotide Z. For example, the term “IL-1RN(+2018) allele” refers to alternative forms of the IL-1RN gene at marker+2018. “IL-1RN (+2018) allele 1” refers to a form of the IL-1RN genewhich contains a cytosine (C) at position +2018 of the sense strand.Clay et al., Hum. Genet. 97:723-26, 1996. “IL-1RN (+2018) allele 2”refers to a form of the IL-1RN gene which contains a thymine (T) atposition +2018 of the plus strand. When a subject has two identicalIL-1RN alleles, the subject is said to be homozygous, or to have thehomozygous state. When a subject has two different IL-1RN alleles, thesubject is said to be heterozygous, or to have the heterozygous state.The term “IL-1RN (+2018) allele 2,2” refers to the homozygous IL-1RN(+2018) allele 2 state. Conversely, the term “IL-1RN (+2018) allele 1,1”refers to the homozygous IL-1RN (+2018) allele 1 state. The term “IL-1RN(+2018) allele 1,2” refers to the heterozygous allele 1 and 2 state.

“IL-1 related” as used herein is meant to include all genes related tothe human IL-1 locus genes on human chromosome 2 (2q 12-14). Theseinclude IL-1 genes of the human IL-1 gene cluster located at chromosome2 (2q 13-14) which include: the IL-1A gene which encodes interleukin-1α,the IL-1B gene which encodes interleukin-1β, and the IL-1RN (or IL-1ra)gene which encodes the interleukin-1 receptor antagonist. Furthermorethese IL-1 related genes include the type I and type II human IL-1receptor genes located on human chromosome 2 (2q12) and their mousehomologs located on mouse chromosome 1 at position 19.5 cM.Interleukin-1α, interleukin-1β, and interleukin-1RN are related in somuch as they all bind to IL-1 type I receptors, however onlyinterleukin-1 (and interleukin-10 are agonist ligands which activateIL-1 type I receptors, while interleukin-1RN is a naturally occurringantagonist ligand. Where the term “IL-1” is used in reference to a geneproduct or polypeptide, it is meant to refer to all gene productsencoded by the interleukin-1 locus on human chromosome 2 (2q 12-14) andtheir corresponding homologs from other species or functional variantsthereof. The term IL-1 thus includes secreted polypeptides which promotean inflammatory response, such as IL-1α and IL-1β, as well as a secretedpolypeptide which antagonize inflammatory responses, such as IL-1receptor antagonist and the IL-1 type II (decoy) receptor.

An “IL-1 receptor” or “IL-1R” refers to various cell membrane boundprotein receptors capable of binding to and/or transducing a signal fromIL-1 locus-encoded ligand. The term applies to any of the proteins whichare capable of binding interleukin-1 (IL-1) molecules and, in theirnative configuration as mammalian plasma membrane proteins, presumablyplay a role in transducing the signal provided by IL-1 to a cell. Asused herein, the term includes analogs of native proteins withIL-1-binding or signal transducing activity. Examples include the humanand murine IL-1 receptors described in U.S. Pat. No. 4,968,607. The term“IL-1 nucleic acid” refers to a nucleic acid encoding an IL-1 protein.

An “IL-1 polypeptide” and “IL-1 protein” are intended to encompasspolypeptides comprising the amino acid sequence encoded by the IL-1genomic DNA sequences shown in FIGS. 1, 2, and 3, or fragments thereof,and homologs thereof and include agonist and antagonist polypeptides.

“In-stent stenosis” refers to the progressive occlusion within a stentthat has been placed during angioplasty. In-stent stenosis is a form ofrestenosis that takes place within an arterial stent.

“Increased risk” refers to a statistically higher frequency ofoccurrence of the disease or condition in an individual carrying aparticular polymorphic allele in comparison to the frequency ofoccurrence of the disease or condition in a member of a population thatdoes not carry the particular polymorphic allele.

The term “interact” as used herein is meant to include detectablerelationships or associations (e.g. biochemical interactions) betweenmolecules, such as interactions between protein-protein, protein-nucleicacid, nucleic acid-nucleic acid and protein-small molecule or nucleicacid-small molecule in nature.

The term “isolated” as used herein with respect to nucleic acids, suchas DNA or RNA, refers to molecules separated from other DNAs, or RNAs,respectively, that are present in the natural source of themacromolecule. For example, an isolated nucleic acid encoding one of thesubject IL-1 polypeptides preferably includes no more than 10 kilobases(kb) of nucleic acid sequence which naturally immediately flanks theIL-1 gene in genomic DNA, more preferably no more than 5 kb of suchnaturally occurring flanking sequences, and most preferably less than1.5 kb of such naturally occurring flanking sequence. The term isolatedas used herein also refers to a nucleic acid or peptide that issubstantially free of cellular material, viral material, or culturemedium when produced by recombinant DNA techniques, or chemicalprecursors or other chemicals when chemically synthesized. Moreover, an“isolated nucleic acid” is meant to include nucleic acid fragments whichare not naturally occurring as fragments and would not be found in thenatural state. The term “isolated” is also used herein to refer topolypeptides which are isolated from other cellular proteins and ismeant to encompass both purified and recombinant polypeptides.

A “knock-in” transgenic animal refers to an animal that has had amodified gene introduced into its genome and the modified gene can be ofexogenous or endogenous origin.

A “knock-out” transgenic animal refers to an animal in which there ispartial or complete suppression of the expression of an endogenous gene(e.g, based on deletion of at least a portion of the gene, replacementof at least a portion of the gene with a second sequence, introductionof stop codons, the mutation of bases encoding critical amino acids, orthe removal of an intron junction, etc.).

A “knock-out construct” refers to a nucleic acid sequence that can beused to decrease or suppress expression of a protein encoded byendogenous DNA sequences in a cell. In a simple example, the knock-outconstruct is comprised of a gene, such as the IL-1RN gene, with adeletion in a critical portion of the gene so that active protein cannotbe expressed therefrom. Alternatively, a number of termination codonscan be added to the native gene to cause early termination of theprotein or an intron junction can be inactivated. In a typical knock-outconstruct, some portion of the gene is replaced with a selectable marker(such as the neo gene) so that the gene can be represented as follows:IL-1RN 5′/neo/IL-1RN 3′, where IL-1RN5′ and IL-1RN 3′, refer to genomicor cDNA sequences which are, respectively, upstream and downstreamrelative to a portion of the IL-1RN gene and where neo refers to aneomycin resistance gene. In another knock-out construct, a secondselectable marker is added in a flanking position so that the gene canbe represented as: IL-1RN/neo/IL-1RN/TK, where TK is a thymidine kinasegene which can be added to either the IL-1RN5′ or the IL-1RN3′ sequenceof the preceding construct and which further can be selected against(i.e. is a negative selectable marker) in appropriate media. Thistwo-marker construct allows the selection of homologous recombinationevents, which removes the flanking TK marker, from non-homologousrecombination events which typically retain the TK sequences. The genedeletion and/or replacement can be from the exons, introns, especiallyintron junctions, and/or the regulatory regions such as promoters.

“Linkage disequilibrium” refers to co-inheritance of two alleles atfrequencies greater than would be expected from the separate frequenciesof occurrence of each allele in a given control population. The expectedfrequency of occurrence of two alleles that are inherited independentlyis the frequency of the first allele multiplied by the frequency of thesecond allele. Alleles that co-occur at expected frequencies are said tobe in “linkage equilibrium”. The cause of linkage disequilibrium isoften unclear. It can be due to selection for certain allelecombinations or to recent admixture of genetically heterogeneouspopulations. In addition, in the case of markers that are very tightlylinked to a disease gene, an association of an allele (or group oflinked alleles) with the disease gene is expected if the diseasemutation occurred in the recent past, so that sufficient time has notelapsed for equilibrium to be achieved through recombination events inthe specific chromosomal region. When referring to allelic patterns thatare comprised of more than one allele, a first allelic pattern is inlinkage disequilibrium with a second allelic pattern if all the allelesthat comprise the first allelic pattern are in linkage disequilibriumwith at least one of the alleles of the second allelic pattern. Anexample of linkage disequilibrium is that which occurs between thealleles at the IL-1RN (+2018) and IL-1RN (VNTR) polymorphic sites. Thetwo alleles at IL-1RN (+2018) are 100% in linkage disequilibrium withthe two most frequent alleles of IL-1RN (VNTR), which are allele 1 andallele 2.

The term “marker” refers to a sequence in the genome that is known tovary among individuals. For example, the IL-1RN gene has a marker thatconsists of a variable number of tandem repeats (VNTR).

“Modulate” refers to the ability of a substance to regulate bioactivity.When applied to an IL-1 bioactivity, an agonist or antagonist canmodulate bioactivity for example by agonizing or antagonizing an IL-1synthesis, receptor interaction, or IL-1 mediated signal transductionmechanism.

A “mutated gene” or “mutation” or “functional mutation” refers to anallelic form of a gene, which is capable of altering the phenotype of asubject having the mutated gene relative to a subject which does nothave the mutated gene. The altered phenotype caused by a mutation can becorrected or compensated for by certain agents. If a subject must behomozygous for this mutation to have an altered phenotype, the mutationis said to be recessive. If one copy of the mutated gene is sufficientto alter the phenotype of the subject, the mutation is said to bedominant. If a subject has one copy of the mutated gene and has aphenotype that is intermediate between that of a homozygous and that ofa heterozygous subject (for that gene), the mutation is said to beco-dominant.

A “non-human animal” of the invention includes mammals such as rodents,non-human primates, sheep, dogs, cows, goats, etc. Preferred non-humananimals are selected from the rodent family including rat and mouse,most preferably mouse, though transgenic amphibians, such as members ofthe Xenopus genus, and transgenic chickens can also provide importanttools for understanding and identifying agents which can affect, forexample, embryogenesis and tissue formation. The term “chimeric animal”is used herein to refer to animals in which the recombinant gene isfound, or in which the recombinant gene is expressed in some but not allcells of the animal. The term “tissue-specific chimeric animal”indicates that one of the recombinant IL-1 genes is present and/orexpressed or disrupted in some tissues but not others. The term“non-human mammal” refers to any members of the class Mammalia, exceptfor humans.

As used herein, the term “nucleic acid” refers to polynucleotides oroligonucleotides such as deoxyribonucleic acid (DNA), and, whereappropriate, ribonucleic acid (RNA). The term should also be understoodto include, as equivalents, analogs of either RNA or DNA made fromnucleotide analogs (e.g. peptide nucleic acids) and as applicable to theembodiment being described, single (sense or antisense) anddouble-stranded polynucleotides.

“Occlusive disorder” refers to that cardiovascular disordercharacterized by the progressive thickening of an arterial wall,associated with the presence of an atherosclerotic intimal lesion withinan artery. Occlusive disorder leads to progressive blockage of theartery. With sufficient progression, the occlusive disorder can reduceflow in the artery to the point that clinical signs and symptoms areproduced in the tissues perfused by the artery. These clinical eventsrelate to ischemia of the perfused tissues. When severe, ischemia isaccompanied by tissue death, called infarction or gangrene. Occlusivedisorder is associated with the allele pattern 2s at the IL-1 locus.

An “occlusive disorder therapeutic” refers to any agent or therapeuticregimen (including pharmaceuticals, nutraceuticals and surgical means)that prevents or postpones the development of or reduces the extent ofan abnormality constitutive of an occlusive disorder in a subject.Examples of occlusive disorder therapeutics include those agents thatare anti-oxidants, those that lower serum lipids, those that block theaction of oxidized lipids and other agents that influence lipidmetabolism or otherwise have lipid-active effects.

A “peripheral vascular disease” (“PVD”) is a cardiovascular diseaseresulting from the blockage of the peripheral (i.e., non-coronary)arteries. Blockage can occur suddenly, by mechanisms such as plaquerupture or embolization, as occurs in fragile plaque disease. Blockagecan occur progressively, with narrowing of the artery via myointimalhyperplasia and plaque formation, as in occlusive disease. Blockage canbe complete or partial. Those clinical signs and symptoms resulting fromthe blockage of peripheral arteries are manifestations of peripheralvascular disease. Manifestations of peripheral vascular diseasesinclude, inter alia, claudication, ischemia, intestinal angina,vascular-based renal insufficiency, transient ischemic attacks, aneurysmformation, peripheral embolization and stroke. Ischemic cerebrovasculardisease is a type of peripheral vascular disease. The term“polymorphism” refers to the coexistence of more than one form of a geneor portion (e.g., allelic variant) thereof. A portion of a gene of whichthere are at least two different forms, i.e., two different nucleotidesequences, is referred to as a “polymorphic region of a gene”. Aspecific genetic sequence at a polymorphic region of a gene is anallele. A polymorphic region can be a single nucleotide, the identity ofwhich differs in different alleles. A polymorphic region can also beseveral nucleotides long.

The term “propensity to disease,” also “predisposition” or“susceptibility” to disease or any similar phrase, means that certainalleles are hereby discovered to be associated with or predictive ofILD. The alleles are thus over-represented in frequency in individualswith disease as compared to healthy individuals. Thus, these alleles canbe used to predict disease even in pre-symptomatic or pre-diseasedindividuals.

The term “restenosis” refers to any preocclusive lesion that developsfollowing a reconstructive procedure in a diseased blood vessel. Theterm is not only applied to the recurrence of a pre-existing stenosis,but also to previously normal vessels such as vein grafts that becomepartially occluded following vascular bypass. Restenosis refers to anyluminal narrowing that occurs following an injury to the vessel wall.Injuries resulting in restenosis can therefore include trauma to anatherosclerotic lesion (as seen with angioplasty), a resection of alesion (as seen with endarterectomy), an external trauma (e.g., across-clamping injury), or a surgical anastomosis. Restenosis typicallyresults from a hyperplasia.

Restenosis can occur as the result of any kind of vascularreconstruction, whether in the coronary vasculature or in the periphery(Colbum and Moore (1998) Myointimal Hyperplasia pp. 690-709 in VascularSurgery: A Comprehensive Review (Philadelphia: Saunders, 1998)). Forexample, studies have reported symptomatic restenosis rates of 30-50%following coronary angioplasties (see Berk and Harris (1995) Adv.Intern. Med. 40:455-501). After carotid endarterectomies, as a furtherexample, 20% of patients studied had a luminal narrowing greater than50% (Clagett et al. (1986) J. Vasc. Surg. 3:10-23). Yet another exampleof restenosis is seen in infrainguinal vascular bypasses, where 40-60%of prosthetic grafts and 20-40% of the vein grafts are occluded at threeyears (Dalman and Taylor (1990) Ann. Vasc. Surg. 3:109-312, Szilagyi etal. (1973) Ann. Surg. 178:232-246). Different degrees of symptomatologyaccompany preocclusive lesions in different anatomical locations, due toa combination of factors including the different calibers of the vesselsinvolved, the extent of residual disease and local hemodynamics.

A “restenosis associated allele” refers to an allele whose presence in asubject indicates that the subject has or is susceptible to developing arestenosis. Examples of restenosis associated alleles include allele 1of the +4845 marker of IL-1A; allele 1 of the +3954 marker of IL-1B;allele 1 of the −511 markerofIL-1B; and allele 1 of the +2018markerofIL-1RN. Still other linked polymorphic loci associated withrestenosis include: the IL-1RN (VNTR) polymorphism, the IL-1RN gene+1731 polymorphism; the IL-1RN gene +1812 polymorphism; the IL-1RN gene+1868 polymorphism; the IL-1RN gene +1887 polymorphism; the IL-1RN+8006polymorphism, the IL-1RN+8061 polymorphism, the IL-1B −31 polymorphismand the IL-1B −511 polymorphism. Other restenosis associated allelesthat have been described in the art include certain alleles inangiotensin converting enzymes (See e.g. Kasi et al., (1996) Am. J.Cardiol 77: 875-77).

A “restenosis causative functional mutation” refers to a mutation whichcauses or contributes to the development of restenosis in a subject.Preferred mutations occur within the IL-1 complex. A restenosiscausative functional mutation occurring within an IL-1 gene (e.g. IL-1A,IL-1B or IL-1RN) or a gene locus, which is linked thereto, may alter,for example, the open reading frame or splicing pattern of the gene,thereby resulting in the formation of an inactive or hypoactive geneproduct. For example, a mutation which occurs in intron 6 of the IL-1Alocus corresponds to a variable number of tandem repeat 46 bp sequencescorresponding to from five to 18 repeat units (Bailly, et al. (1993)Eur. J. Immunol. 23: 1240-45). These repeat sequences contain threepotential binding sites for transcriptional factors: an SP1 site, aviral enhancer element, and a glucocorticoid-responsive element;therefore individuals carrying IL-1A intron 6 VNTR alleles with largenumbers of repeat units may be subject to altered transcriptionalregulation of the IL-1A gene and consequent perturbations ofinflammatory cytokine production. Indeed, there is evidence thatincreased repeat number at this polymorphic IL-1A locus leads todecreased IL-1α synthesis (Bailly et al. (1996) Mol Immunol 33:999-1006). Alternatively, a mutation can result in a hyperactive geneproduct. For example, allele 2 of the IL-1β (G at +6912) polymorphismoccurs in the 3′ UTR (untranslated region) of the IL-1B mRNA and isassociated with an approximately four-fold increase in the steady statelevels of both IL-1B mRNA and IL-1B protein compared to those levelsassociated with allele 1 of the IL-1B gene (© at +6912). Further, anIL-1B (−511) mutation occurs near a promoter binding site for a negativeglucocorticoid response element (Zhang et al. (1997) DNA Cell Biol 16:145-52). This element potentiates a four-fold repression of IL-1Bexpression by dexamethosone and a deletion of this negative responseelements causes a 2.5-fold increase in IL-1B promoter activity. TheIL-1B (−511) polymorphism may thus directly affect cytokine productionand inflammatory responses. These examples demonstrate that geneticvariants occurring in the IL-1A or IL-1B gene can directly lead to thealtered production or regulation of IL-1 cytokine activity.

A “restenosis therapeutic” refers to any agent or therapeutic regimen(including pharmaceuticals, nutraceuticals and surgical means) thatprevents or postpones the development of or alleviates the symptoms of arestenosis in a subject. A restenosis therapeutic can be a polypeptide,peptidomimetic, nucleic acid or other inorganic or organic molecule,preferably a “small molecule” including vitamins, minerals and othernutrients. Preferably a restenosis therapeutic can modulate at least oneactivity of an IL-1 polypeptide, e.g., interaction with a receptor, bymimicking or potentiating (agonizing) or inhibiting (antagonizing) theeffects of a naturally-occurring polypeptide. An agonist can be awild-type protein or derivative thereof having at least one bioactivityof the wild-type, e.g., receptor binding activity. An agonist can alsobe a compound that upregulates expression of a gene or which increasesat least one bioactivity of a protein. An agonist can also be a compoundwhich increases the interaction of a polypeptide with another molecule,e.g., a receptor. An antagonist can be a compound which inhibits ordecreases the interaction between a protein and another molecule, e.g.,a receptor or an agent that blocks signal transduction orpost-translation processing (e.g., IL-1 converting enzyme (ICE)inhibitor). Accordingly, a preferred antagonist is a compound whichinhibits or decreases binding to a receptor and thereby blockssubsequent activation of the receptor. An antagonist can also be acompound that downregulates expression of a gene or which reduces theamount of a protein present. The antagonist can be a dominant negativeform of a polypeptide, e.g., a form of a polypeptide which is capable ofinteracting with a target peptide, e.g., a receptor, but which does notpromote the activation of the receptor. The antagonist can also be anucleic acid encoding a dominant negative form of a polypeptide, anantisense nucleic acid, or a ribozyrne capable of interactingspecifically with an RNA. Yet other antagonists are molecules which bindto a polypeptide and inhibit its action. Such molecules includepeptides, e.g., forms of target peptides which do not have biologicalactivity, and which inhibit binding to receptors. Thus, such peptideswill bind to the active site of a protein and prevent it frominteracting with target peptides. Yet other antagonists includeantibodies that specifically interact with an epitope of a molecule,such that binding interferes with the biological function of thepolypeptide. In yet another preferred embodiment, the antagonist is asmall molecule, such as a molecule capable of inhibiting the interactionbetween a polypeptide and a target receptor. Alternatively, the smallmolecule can function as an antagonist by interacting with sites otherthan the receptor binding site. Preferred restenosis therapeuticsinclude agents that suppress the development of neointimal hyperplasia,including lipid lowering drugs, antiplatelet agents, anti-inflammatoryagents, antihypertensive agents and anticoagulants; and agents thatdirectly inhibit cellular growth. Furthermore, surgical decisions at thetime of the primary procedure or at the time of a secondary surgicaloperation could differ depending on whether the patient was at higherrisk for a more prolific inflammation-mediated injury response. Thedecision to employ a stent as part of an endovascular procedure could begoverned, for example, by an awareness of a patient's higher risk formore aggressive vascular response to injury.

A “risk factor” is a factor identified to be associated with anincreased risk. A risk factor for a cardiovascular disorder or acardiovascular disease is any factor identified to be associated with anincreased risk of developing those conditions or of worsening thoseconditions. A risk factor can also be associated with an increased riskof an adverse clinical event or an adverse clinical outcome in a patientwith a cardiovascular disorder. Risk factors for cardiovascular diseaseinclude smoking, adverse lipid profiles, elevated lipids or cholesterol,diabetes, hypertension, hypercoagulable states, elevated homocysteinelevels, and lack of exercise. Carrying a particular polymorphic alleleis a risk factor for a particular cardiovascular disorder, and isassociated with an increased risk of the particular disorder.

“Small molecule” as used herein, is meant to refer to a composition,which has a molecular weight of less than about 5 kD and most preferablyless than about 4 kD. Small molecules can be nucleic acids, peptides,peptidomimetics, carbohydrates, lipids or other organic or inorganicmolecules.

As used herein, the term “specifically hybridizes” or “specificallydetects” refers to the ability of a nucleic acid molecule to hybridizeto at least approximately 6 consecutive nucleotides of a sample nucleicacid.

“Transcriptional regulatory sequence” is a generic term used throughoutthe specification to refer to DNA sequences, such as initiation signals,enhancers, and promoters, which induce or control transcription ofprotein coding sequences with which they are operably linked.

As used herein, the term “transgene” means a nucleic acid sequence(encoding, e.g., one of the IL-1 polypeptides, or an antisensetranscript thereto) which has been introduced into a cell. A transgenecould be partly or entirely heterologous, i.e., foreign, to thetransgenic animal or cell into which it is introduced, or, is homologousto an endogenous gene of the transgenic animal or cell into which it isintroduced, but which is designed to be inserted, or is inserted, intothe animal's genome in such a way as to alter the genome of the cellinto which it is inserted (e.g., it is inserted at a location whichdiffers from that of the natural gene or its insertion results in aknockout). A transgene can also be present in a cell in the form of anepisome. A transgene can include one or more transcriptional regulatorysequences and any other nucleic acid, such as introns, that may benecessary for optimal expression of a selected nucleic acid.

A “transgenic animal” refers to any animal, preferably a non-humanmammal, bird or an amphibian, in which one or more of the cells of theanimal contain heterologous nucleic acid introduced by way of humanintervention, such as by transgenic techniques well known in the art.The nucleic acid is introduced into the cell, directly or indirectly byintroduction into a precursor of the cell, by way of deliberate geneticmanipulation, such as by microinjection or by infection with arecombinant virus. The term genetic manipulation does not includeclassical cross-breeding, or in vitro fertilization, but rather isdirected to the introduction of a recombinant DNA molecule. Thismolecule may be integrated within a chromosome, or it may beextrachromosomally replicating DNA. In the typical transgenic animalsdescribed herein, the transgene causes cells to express a recombinantform of one of an IL-1 polypeptide, e.g. either agonistic orantagonistic forms. However, transgenic animals in which the recombinantgene is silent are also contemplated, as for example, the FLP or CRErecombinase dependent constructs described below. Moreover, “transgenicanimal” also includes those recombinant animals in which gene disruptionof one or more genes is caused by human intervention, including bothrecombination and antisense techniques. The term is intended to includeall progeny generations. Thus, the founder animal and all F1, F2, F3,and so on, progeny thereof are included.

The term “treating” as used herein is intended to encompass curing aswell as ameliorating at least one symptom of a disease or at least oneabnormality associated with a disorder. Treating a cardiovasculardisorder can take place by administering a cardiovascular disordertherapeutic. Treating a cardiovascular disorder can also take place bymodifying risk factors that are related to the cardiovascular disorder.

A “treatment plan” refers to at least one intervention undertaken tomodify the effect of a risk factor upon a patient. A treatment plan fora cardiovascular disorder or disease can address those risk factors thatpertain to cardiovascular disorders or diseases. A treatment plan caninclude an intervention that focuses on changing patient behavior, suchas stopping smoking. A treatment plan can include an interventionwhereby a therapeutic agent is administered to a patient. As examples,cholesterol levels can be lowered with proper medication, and diabetescan be controlled with insulin. Nicotine addiction can be treated bywithdrawal medications. A treatment plan can include an interventionthat is diagnostic. The presence of the risk factor of hypertension, forexample, can give rise to a diagnostic intervention whereby the etiologyof the hypertension is determined. After the reason for the hypertensionis identified, further treatments may be administered.

The term “vector” refers to a nucleic acid molecule, which is capable oftransporting another nucleic acid to which it has been linked. One typeof preferred vector is an episome, i.e., a nucleic acid capable ofextra-chromosomal replication. Preferred vectors are those capable ofautonomous replication and/or expression of nucleic acids to which theyare linked. Vectors capable of directing the expression of genes towhich they are operatively linked are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of “plasmids” which refer generally tocircular double stranded DNA loops which, in their vector form are notbound to the chromosome. In the present specification, “plasmid” and“vector” are used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors which serve equivalent functions andwhich become known in the art subsequently hereto.

The term “wild-type allele” refers to an allele of a gene which, whenpresent in two copies in a subject results in a wild-type phenotype.There can be several different wild-type alleles of a specific gene,since certain nucleotide changes in a gene may not affect the phenotypeof a subject having two copies of the gene with the nucleotide changes.

4.2 Predictive Medicine

4.2.1. Polymorphisms Associated with Restenosis

The present invention is based at least in part, on the identificationof alleles that are associated (to a statistically significant extent)with the development of a restenosis in subjects. Therefore, detectionof these alleles, alone or in conjunction with another means in asubject indicate that the subject has or is predisposed to thedevelopment of a restenosis. For example, IL-1 polymorphic alleles whichare associated with a propensity for developing restenosis includeallele 1 of each of the following markers: IL-1A (+4845), IL-1B (+3954),IL-1B (−511), IL-1RN (+2018) and IL-1RN (VNTR) or an allele that is inlinkage disequilibrium with one of the aforementioned alleles. Inparticularly preferred embodiments, the presence of a particular allelicpattern of one or more of the abovementioned IL-1 polymorphic loci isused to predict the susceptibility of an individual to developingrestenosis. In particular, there are three patterns of alleles at fourpolymorphic loci in the IL-1 gene cluster that show various associationswith particular cardiovascular disorders. These patterns are referred toherein as patterns 1, 2 and 3. Pattern 1 comprises an allelic patternincluding allele 2 of IL-1A (+4845) or IL-1B (+3954) and allele 1 ofIL-1B (−511) or IL-1RN (+2018), or an allele that is in linkagedisequilibrium with one of the aforementioned allele. In a preferredembodiment, this allelic pattern permits the diagnosis of fragile plaquedisorder. Pattern 2 comprises an allelic pattern including allele 2 ofIL-1B (−511) or IL-1RN (+2018) and allele 1 of IL-1A (+4845) or IL-1B(+3954), or an allele that is in linkage disequilibrium with one of theaforementioned alleles. In a preferred embodiment, this allelic patternpermits the diagnosis of occlusive cardiovascular disorder. Pattern 3comprises an allelic pattern including allele 1 of IL-1A (+4845) orallele 1 of IL-1B (+3954), and allele 1 of IL-1B (−511) or allele 1 ofIL-1RN (+2018), or an allele that is in linkage disequilibrium with oneof the aforementioned alleles. In a preferred embodiment, this allelicpattern permits the diagnosis of a restenosis disorder

These IL-1 locus polymorphisms represent single base variations withinthe IL-1A/IL-1B/IL-1RN gene cluster (see FIG. 4). The IL-1A (+4845)polymorphism is a single base variation (allele 1 is G, allele 2 is T)at position +4845 within Exon V of the IL-1A gene which encodes theinflammatory cytokine IL-1a (Gubler, et al.(1989) Interleukin,inflammation and disease (Bomford and Henderson, eds.) p. 31-45,Elsevier publishers; and Van den velden and Reitsma (1993) Hum MolGenetics 2:1753-50). The IL-1A (+4845) polymorphism occurs in the codingregion of the gene and results in a single amino acid variation in theencoded protein (Van den Velden and Reitsma (1993) Hum Mol Genet.2:1753). The IL-1B (−511) polymorphism is a single base pair variation(allele 1 is C, allele 2 is T) which occurs 511 base pairs upstream ofthe site of IL-1B gene transcription initiation (Di Giovine et al.(1992) Hum Mol Genet. 1: 450). The IL-1B (+3954) polymorphism was firstdescribed as a Taq I restriction fragment length polymorphism (RFLP)(Pociot et al. (1992) Eur J Clin Invest 22: 396-402) and hassubsequently been characterized as a single base variation (allele 1 isC, allele 2 is T) at position +3954 in Exon V of the IL-1B gene (diGiovine et al. (1995) Cytokine 7: 600-606). This single nucleotidechange in the open reading frame of IL-1B does not appear toqualitatively affect the sequence of the encoded IL-1 beta polypeptidebecause it occurs at the third position of a TTC phenylalanine codon (F)of allele 1 and therefore allele 2 merely substitutes a TT phenylalaninecodon at this position which encodes amino acid 105 of the IL-1B geneproduct. Finally, the IL-RN variable number of tandem repeats (VNTR)polymorphism occurs within the second intron the IL-1 receptorantagonist encoding gene (Steinkasserer (1991) Nucleic Acids Res 19:5090-5). Allele 2 of the of the IL-1RN (VNTR) polymorphism correspondsto two repeats of an 86-base pair sequence, while allele 1 correspondsto four repeats, allele 3 to three repeats, allele 4 to five repeats,and allele 5 to six repeats (Tarlow et al. (1993) Hum Genet. 91: 403-4).Detection of any one of these IL-1 allelic variants in an individualsuggests an increased likelihood of developing restenosis in comparisonto a control individual who does not carry the allele 2 variant at thesame locus.

However, because these alleles are in linkage disequilibrium with otheralleles, the detection of such other linked alleles can also indicatethat the subject has or is predisposed to the development of arestenosis. For example, the following alleles of the IL-1 (33221461)haplotype are in linkage disequilibrium:

allele 3 of the 222/223 marker of IL-1A allele 3 of the gz5/gz6 markerof IL-1A allele 2 of the −889 marker of IL-1A allele 2 of the +3954marker of IL-1B allele 1 of the −511 marker of IL-1B allele 4 of thegaat.p33330 marker allele 6 of the Y31 marker allele 1 of the VNTR or(+2018) marker of IL-1RN

Therefore, allele 1 of IL-1B (−511) and allele 1 of IL-1RN (VNTR) are instrong linkage disequilibrium with one another and each of these is inlinkage disequilibrium with allele 1 of the −511 marker of IL-1B.Furthermore, in alternative embodiments of the present invention,genotyping analysis at the 222/223 marker of IL-1A, the gz5/gz6 markerof IL-1A, the −889 marker of IL-1A, the +3954 marker of IL-1B, thegaat.p33330 marker of the IL-1B/IL-1RN intergenic region, or the Y31marker of the IL-1B/IL-1RN intergenic region is determined, and thepresence of a polymorphic allele which is linked to one or more of thepreferred restenosis-predictive alleles is detected.

In addition, allele 1 of the IL-1RN (+2018) polymorphism (Clay et al.(1996) Hum Genet. 97: 723-26), also referred to as exon 2 (8006)(GenBank:X64532 at 8006) is known to be in linkage disequilibrium withallele 1 of the IL-1RN (VNTR) polymorphic locus, which in turn is a partof the 33221461 human haplotype. In contrast, allele 2 of the IL-1RN(+2018) locus (i.e. C at +2018), is an allelic variant associated withthe 44112332 haplotype and allele 2 of the IL-1RN (VNTR) polymorphiclocus. The IL-1RN (VNTR) therefore provides an alternative target forprognostic genotyping analysis to determine an individual's likelihoodof developing restenosis. Similarly, three other polymorphisms in anIL-1RN alternative exon (Exon 1ic, which produces an intracellular formof the gene product) are also in linkage disequilibrium with allele 2 ofIL-1RN (VNTR) (Clay et al. (1996) Hum Genet. 97: 723-26). These include:the IL-1RN exon 1ic (1812) polymorphism (GenBank:X77090 at 1812); theIL-1RN exon 1ic (1868) polymorphism (GenBank:X77090 at 1868); and theIL-1RN exon 1ic (1887) polymorphism (GenBank:X77090 at 1887).Furthermore yet another polymorphism in the promoter for thealternatively spliced intracellular form of the gene, the Pic (1731)polymorphism (GenBank:X77090 at 1731), is also in linkage disequilibriumwith allele 2 of the IL-1RN (VNTR) polymorphic locus (Clay et al. (1996)Hum Genet. 97: 723-26). The corresponding sequence alterations for eachof these IL-1RN polymorphic loci is shown below.

Exon 1ic-1 Exon 1ic-2 Exon 1ic-3 Pic Exon 2 (1812 of (1868 of (1887 of(1731 of (+2018 of GB: GB: GB: GB: Allele # IL-1RN) X77090) X77090X77090) X77090) 1 T G A G G 2 C A G C AFor each of these polymorphic loci, the allele 1 sequence variant hasbeen determined to be in linkage disequilibrium with allele 1 of theIL-1RN (VNTR) locus (Clay et al. (1996) Hum Genet. 97: 723-26).

Further, allele 1 of IL-1B (+3954), which has been pointed out as aprognostic indicator of an increased propensity for developingrestenosis is a component of a second haplotype, the 44112332 haplotypeof co-inherited IL-1 locus polymorphic alleles (Cox, et al. (1998) Am.J. Hum. Genet. 62: 1180-88). Specifically, the 44112332 haplotypecomprises the following genotype:

allele 4 of the 222/223 marker of IL-1A allele 4 of the gz5/gz6 markerof IL-1A allele 1 of the −889 marker of IL-1A allele 1 of the +3954marker of IL-1B allele 2 of the −511 marker of IL-1B allele 3 of thegaat.p33330 marker allele 3 of the Y31 marker allele 2 of the VNTRmarker of IL-1RN

In addition to the allelic patterns described above, as describedherein, one of skill in the art can readily identify other alleles(including polymorphisms and mutations) that are in linkagedisequilibrium with an allele associated with restenosis. For example, anucleic acid sample from a first group of subjects without restenosiscan be collected, as well as DNA from a second group of subjects withrestenosis. The nucleic acid sample can then be compared to identifythose alleles that are over-represented in the second group as comparedwith the first group, wherein such alleles are presumably associatedwith restenosis. Alternatively, alleles that are in linkagedisequilibrium with a restenosis associated allele can be identified,for example, by genotyping a large population and performing statisticalanalysis to determine which alleles appear more commonly together thanexpected Preferably the group is chosen to be comprised of geneticallyrelated individuals. Genetically related individuals include individualsfrom the same race, the same ethnic group, or even the same family. Asthe degree of genetic relatedness between a control group and a testgroup increases, so does the predictive value of polymorphic alleleswhich are ever more distantly linked to a disease-causing allele. Thisis because less evolutionary time has passed to allow polymorphismswhich are linked along a chromosome in a founder population toredistribute through genetic cross-over events Thus race-specific,ethnic-specific, and even family-specific diagnostic genotyping assayscan be developed to allow for the detection of disease alleles whicharose at ever more recent times in human evolution, e.g., afterdivergence of the major human races, after the separation of humanpopulations into distinct ethnic groups, and even within the recenthistory of a particular family line.

Linkage disequilibrium between two polymorphic markers or between onepolymorphic marker and a disease-causing mutation is a meta-stable stateAbsent selective pressure or the sporadic linked reoccurrence of theunderlying mutational events, the polymorphisms will eventually becomedisassociated by chromosomal recombination events and will thereby reachlinkage equilibrium through the course of human evolution. Thus, thelikelihood of finding a polymorphic allele in linkage disequilibriumwith a disease or condition may increase with changes in at least twofactors: decreasing physical distance between the polymorphic marker andthe disease-causing mutation, and decreasing number of meioticgenerations available for the dissociation of the linked pair.Consideration of the latter factor suggests that, the more closelyrelated two individuals are, the more likely they will share a commonparental chromosome or chromosomal region containing the linkedpolymorphisms and the less likely that this linked pair will have becomeunlinked through meiotic cross-over events occurring each generation. Asa result, the more closely related two individuals are, the more likelyit is that widely spaced polymorphisms may be co-inherited. Thus, forindividuals related by common race, ethnicity or family, the reliabilityof ever more distantly spaced polymorphic loci can be relied upon as anindicator of inheritance of a linked disease-causing mutation.

Appropriate probes may be designed to hybridize to a specific gene ofthe IL-1 locus, such as IL-1A, IL-1B or IL-1RN or a related gene. Thesegenomic DNA sequences are shown in FIGS. 1, 2 and 3, respectively, andfurther correspond to formal SEQ ID Nos. 15, 16 and 17, respectively.Alternatively, these probes may incorporate other regions of therelevant genomic locus, including intergenic sequences. Indeed the IL-1region of human chromosome 2 spans some 400,000 base pairs and, assumingan average of one single nucleotide polymorphism every 1,000 base pairs,includes some 400 SNPs loci alone. Yet other polymorphisms available foruse with the immediate invention are obtainable from various publicsources. For example, the human genome database collects intragenicSNPs, is searchable by sequence and currently contains approximately2,700 entries (http://hgbase.interactiva.de). Also available is a humanpolymorphism database maintained by the Massachusetts Institute ofTechnology (MIT SNP database(http://www.genome.wi.mit.edu/SNP/human/index.html)). From such sourcesSNPs as well as other human polymorphisms may be found.

For example, examination of the IL-1 region of the human genome in anyone of these databases reveals that the IL-1 locus genes are flanked bya centromere proximal polymorphic marker designated microsatellitemarker AFM220ze3 at 127.4 cM (centiMorgans) (see GenBank Acc. No.Z17008) and a distal polymorphic marker designated microsatellite anchormarker AFMO87xal at 127.9 cM (see GenBank Ace. No. Z16545). These humanpolymorphic loci are both CA dinucleotide repeat microsatellitepolymorphisms, and, as such, show a high degree of heterozygosity inhuman populations. For example, one allele of AFM220ze3 generates a 211bp PCR amplification product with a 5′ primer of the sequenceTGTACCTAAGCCCACCCTT-TAGAGC (SEQ ID No. 18) and a 3′ primer of thesequence TGGCCTCCAGAAACCTCCAA (SEQ ID No. 19). Furthermore, one alleleof AFMO87xal generates a 177 bp PCR amplification product with a 5′primer of the sequence GCTGATATTCTGGTGGGAAA (SEQ ID No.20) and a 3′primer of the sequence GGCAAGAGCAAAACTCTGTC (SEQ ID No. 21). Equivalentprimers corresponding to unique sequences occurring 5′ and 3′ to thesehuman chromosome 2 CA dinucleotide repeat polymorphisms will be apparentto one of skill in the art. Reasonable equivalent primers include thosewhich hybridize within about 1 kb of the designated primer, and whichfurther are anywhere from about 17 bp to about 27 bp in length. Ageneral guideline for designing primers for amplification of uniquehuman chromosomal genomic sequences is that they possess a meltingtemperature of at least about 50° C., wherein an approximate meltingtemperature can be estimated using the formula

T _(melt)=[2×(# of A or T)+4×(# of G or C)].

A number of other human polymorphic loci occur between these two CAdinucleotide repeat polymorphisms and provide additional targets fordetermination of a restenosis prognostic allele in a family or othergroup of genetically related individuals. For example, the NationalCenter for Biotechnology Information web site(www.ncbi.nlm.nih.gov/genemap/) lists a number of polymorphism markersin the region of the IL-1 locus and provides guidance in designingappropriate primers for amplification and analysis of these markers.

Accordingly, the nucleotide segments of the invention may be used fortheir ability to selectively form duplex molecules with complementarystretches of human chromosome 2 q 12-13 or cDNAs from that region or toprovide primers for amplification of DNA or cDNA from this region. Thedesign of appropriate probes for this purpose requires consideration ofa number of factors. For example, fragments having a length of between10, 15, or 18 nucleotides to about 20, or to about 30 nucleotides, willfind particular utility. Longer sequences, e.g., 40, 50, 80, 90, 100,even up to full length, are even more preferred for certain embodiments.Lengths of oligonucleotides of at least about 18 to 20 nucleotides arewell accepted by those of skill in the art as sufficient to allowsufficiently specific hybridization so as to be useful as a molecularprobe. Furthermore, depending on the application envisioned, one willdesire to employ varying conditions of hybridization to achieve varyingdegrees of selectivity of probe towards target sequence. Forapplications requiring high selectivity, one will typically desire toemploy relatively stringent conditions to form the hybrids. For example,relatively low salt and/or high temperature conditions, such as providedby 0.02 M-0.15M NaCl at temperatures of about 50° C. to about 70° C.Such selective conditions may tolerate little, if any, mismatch betweenthe probe and the template or target strand.

Other alleles or other indicia of restenosis can be detected ormonitored in a subject in conjunction with detection of the allelesdescribed above. For example, echocardiography may be performed duringexercise, since studies have found an association between the occurrenceof clinical restenosis and both a positive post-percutaneoustransluminal coronary angioplasty exercise echo as well as high valuesof the pre-surgical wall-motion score index and duration of wall-motionabnormalities (Peters et al. (1997) Circulation 95: 2254-61; Dagianti etal. (1997) Circulation 95: 1176-84; Gentile (1994) Cardiologia 39:651-6). Furthermore, angioscopic studies have shown that the color(yellow versus white) of a patient's arterial plaque is highlypredictive of the occurrence of restenosis following balloon angioplastyin individuals with stable angina (Itoh et al. (1995) Circulation 91:1389-96). In addition, certain polymorphisms in the gene encodingangiotensin converting enzyme have been associated with the occurrenceof restenosis after coronary angioplasty in unstable angina pectoris(See e.g. Kasi et al., (1996) Am J Cardiol 77: 875-77).

In addition, behavioral studies have shown an association betweenhostility and other aspects of a type A behavior pattern and anincreased risk for restenosis following percutaneous transluminalcoronary angioplasty (Goodman et al. (1996) Mayo Clin Proc 71: 729-34).Still other studies have demonstrated an association between variousserum proteins and an increased likelihood of restenosis. For example adrop in the level of antibodies against heat shock protein-65 afterpercutaneous transluminal coronary angioplasty is associated with adecreased risk of developing restenosis relative to individuals in whichno decrease in the level of these antibodies occurred (Mukherjee et al.(1996) Throm Haemost 75: 258-60). Another study has demonstrated anassociation between an elevation of serum amyloid A and the occurrenceof restenosis following angioplasty (Blum et al. (1998) Clin Cardiol 21:655-58). Relatively high levels of plasminogen activator inhibitortype-1 and relatively low levels of plasmin-plasmin inhibitor complexare also associated with restenosis (Ishiwata et al. (1997) Am Heart J133: 387-92), as are high levels of serum lipoprotein A (Hearn et al.(1992) Am J Cardiol 69: 736-39) and elevated levels of monounsaturatedfatty acids (Foley et al. (1992) Cathet Cardiovasc Diagn 25: 25-30).

4.2.2 Detection of Alleles

Many methods are available for detecting specific alleles at humanpolymorphic loci. The preferred method for detecting a specificpolymorphic allele will depend, in part, upon the molecular nature ofthe polymorphism. For example, the various allelic forms of thepolymorphic locus may differ by a single base-pair of the DNA. Suchsingle nucleotide polymorphisms (or SNPs) are major contributors togenetic variation, comprising some 80% of all known polymorphisms, andtheir density in the human genome is estimated to be on average 1 per1,000 base pairs. SNPs are most frequently biallelic-occurring in onlytwo different forms (although up to four different forms of an SNP,corresponding to the four different nucleotide bases occurring in DNA,are theoretically possible). Nevertheless, SNPs are mutationally morestable than other polymorphisms, making them suitable for associationstudies in which linkage disequilibrium between markers and an unknownvariant is used to map disease-causing mutations. In addition, becauseSNPs typically have only two alleles, they can be genotyped by a simpleplus/minus assay rather than a length measurement, making them moreamenable to automation.

A variety of methods are available for detecting the presence of aparticular single nucleotide polymorphic allele in an individual.Advancements in this field have provided accurate, easy, and inexpensivelarge-scale SNP genotyping. Most recently, for example, several newtechniques have been described including dynamic allele-specifichybridization (DASH), microplate array diagonal gel electrophoresis(MADGE), pyrosequencing, oligonucleotide-specific ligation, the TaqMansystem as well as various DNA “chip” technologies such as the AffymetrixSNP chips. These methods require amplification of the target geneticregion, typically by PCR. Still other newly developed methods, based onthe generation of small signal molecules by invasive cleavage followedby mass spectrometry or immobilized padlock probes and rolling-circleamplification, might eventually eliminate the need for PCR. Several ofthe methods known in the art for detecting specific single nucleotidepolymorphisms are summarized below. The method of the present inventionis understood to include all available methods.

Several methods have been developed to facilitate analysis of singlenucleotide polymorphisms. In one embodiment, the single basepolymorphism can be detected by using a specializedexonuclease-resistant nucleotide, as disclosed, e.g., in Mundy, C. R.(U.S. Pat. No. 4,656,127). According to the method, a primercomplementary to the allelic sequence immediately 3′ to the polymorphicsite is permitted to hybridize to a target molecule obtained from aparticular animal or human. If the polymorphic site on the targetmolecule contains a nucleotide that is complementary to the particularexonuclease-resistant nucleotide derivative present, then thatderivative will be incorporated onto the end of the hybridized primer.Such incorporation renders the primer resistant to exonuclease, andthereby permits its detection. Since the identity of theexonuclease-resistant derivative of the sample is known, a finding thatthe primer has become resistant to exonucleases reveals that thenucleotide present in the polymorphic site of the target molecule wascomplementary to that of the nucleotide derivative used in the reaction.This method has the advantage that it does not require the determinationof large amounts of extraneous sequence data.

In another embodiment of the invention, a solution-based method is usedfor determining the identity of the nucleotide of a polymorphic site.Cohen, D. et al. (French Patent 2,650,840; PCT Appln. No. WO91/02087).As in the Mundy method of U.S. Pat. No. 4,656,127, a primer is employedthat is complementary to allelic sequences immediately 3′ to apolymorphic site. The method determines the identity of the nucleotideof that site using labeled dideoxynucleotide derivatives, which, ifcomplementary to the nucleotide of the polymorphic site will becomeincorporated onto the terminus of the primer.

An alternative method, known as Genetic Bit Analysis or GBA™ isdescribed by Goelet, P. et al. (PCT Appln. No. 92/15712). The method ofGoelet, P. et al. uses mixtures of labeled terminators and a primer thatis complementary to the sequence 3′ to a polymorphic site. The labeledterminator that is incorporated is thus determined by, and complementaryto, the nucleotide present in the polymorphic site of the targetmolecule being evaluated. In contrast to the method of Cohen et al.(French Patent 2,650,840; PCT Appln. No. WO91/02087) the method ofGoelet, P. et al. is preferably a heterogeneous phase assay, in whichthe primer or the target molecule is immobilized to a solid phase.

Recently, several primer-guided nucleotide incorporation procedures forassaying polymorphic sites in DNA have been described (Komher, J. S. etal., Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov, B. P., Nucl. AcidsRes. 18:3671 (1990); Syvanen, A.-C., et al., Genomics 8:684-692 (1990);Kuppuswamy, M. N. et al., Proc. Natl. Acad. Sci. (U.S.A.) 88:1143-1147(1991); Prezant, T. R. et al., Hum. Mutat. 1:159-164 (1992); Ugozzoli,L. et al., GATA 9:107-112 (1992); Nyren, P. et al., Anal. Biochem.208:171-175 (1993)). These methods differ from GBA™ in that they allrely on the incorporation of labeled deoxynucleotides to discriminatebetween bases at a polymorphic site. In such a format, since the signalis proportional to the number of deoxynucleotides incorporated,polymorphisms that occur in runs of the same nucleotide can result insignals that are proportional to the length of the run (Syvanen, A.-C.,et al., Amer. J. Hum. Genet. 52:46-59 (1993)).

For mutations that produce premature termination of protein translation,the protein truncation test (PTT) offers an efficient diagnosticapproach (Roest, et. al., (1993) Hum. Mol. Genet. 2:1719-21; van derLuijt, et. al., (1994) Genomics 20:1-4). For PTT, RNA is initiallyisolated from available tissue and reverse-transcribed, and the segmentof interest is amplified by PCR. The products of reverse transcriptionPCR are then used as a template for nested PCR amplification with aprimer that contains an RNA polymerase promoter and a sequence forinitiating eukaryotic translation. After amplification of the region ofinterest, the unique motifs incorporated into the primer permitsequential in vitro transcription and translation of the PCR products.Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis oftranslation products, the appearance of truncated polypeptides signalsthe presence of a mutation that causes premature termination oftranslation. In a variation of this technique, DNA (as opposed to RNA)is used as a PCR template when the target region of interest is derivedfrom a single exon.

Any cell type or tissue may be utilized to obtain nucleic acid samplesfor use in the diagnostics described herein. In a preferred embodiment,the DNA sample is obtained from a bodily fluid, e.g, blood, obtained byknown techniques (e.g. venipuncture) or saliva. Alternatively, nucleicacid tests can be performed on dry samples (e.g. hair or skin). Whenusing RNA or protein, the cells or tissues that may be utilized mustexpress an IL-1 gene.

Diagnostic procedures may also be performed in situ directly upon tissuesections (fixed and/or frozen) of patient tissue obtained from biopsiesor resections, such that no nucleic acid purification is necessary.Nucleic acid reagents may be used as probes and/or primers for such insitu procedures (see, for example, Nuovo, G. J., 1992, PCR in situhybridization: protocols and applications, Raven Press, NY).

In addition to methods which focus primarily on the detection of onenucleic acid sequence, profiles may also be assessed in such detectionschemes. Fingerprint profiles may be generated, for example, byutilizing a differential display procedure, Northern analysis and/orRT-PCR.

A preferred detection method is allele specific hybridization usingprobes overlapping a region of at least one allele of an IL-1proinflammatory haplotype and having about 5, 10, 20, 25, or 30nucleotides around the mutation or polymorphic region. In a preferredembodiment of the invention, several probes capable of hybridizingspecifically to other allelic variants involved in a restenosis areattached to a solid phase support, e.g., a “chip” (which can hold up toabout 250,000 oligonucleotides). Oligonucleotides can be bound to asolid support by a variety of processes, including lithography. Mutationdetection analysis using these chips comprising oligonucleotides, alsotermed “DNA probe arrays” is described e.g., in Cronin et al. (1996)Human Mutation 7:244. In one embodiment, a chip comprises all theallelic variants of at least one polymorphic region of a gene. The solidphase support is then contacted with a test nucleic acid andhybridization to the specific probes is detected. Accordingly, theidentity of numerous allelic variants of one or more genes can beidentified in a simple hybridization experiment.

These techniques may also comprise the step of amplifying the nucleicacid before analysis. Amplification techniques are known to those ofskill in the art and include, but are not limited to cloning, polymerasechain reaction (PCR), polymerase chain reaction of specific alleles(ASA), ligase chain reaction (LCR), nested polymerase chain reaction,self sustained sequence replication (Guatelli, J. C. et al., 1990, Proc.Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system(Kwoh, D. Y. et al., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), andQ-Beta Replicase (Lizardi, P. M. et al., 1988, Bio/Technology 6:1197).

Amplification products may be assayed in a variety of ways, includingsize analysis, restriction digestion followed by size analysis,detecting specific tagged oligonucleotide primers in the reactionproducts, allele-specific oligonucleotide (ASO) hybridization, allelespecific 5′ exonuclease detection, sequencing, hybridization, and thelike.

PCR based detection means can include multiplex amplification of aplurality of markers simultaneously. For example, it is well known inthe art to select PCR primers to generate PCR products that do notoverlap in size and can be analyzed simultaneously. Alternatively, it ispossible to amplify different markers with primers that aredifferentially labeled and thus can each be differentially detected. Ofcourse, hybridization based detection means allow the differentialdetection of multiple PCR products in a sample. Other techniques areknown in the art to allow multiplex analyses of a plurality of markers.

In a merely illustrative embodiment, the method includes the steps of(i) collecting a sample of cells from a patient, (ii) isolating nucleicacid (e.g., genomic, mRNA or both) from the cells of the sample, (iii)contacting the nucleic acid sample with one or more primers whichspecifically hybridize 5′ and 3′ to at least one allele of an IL-1proinflammatory haplotype under conditions such that hybridization andamplification of the allele occurs, and (iv) detecting the amplificationproduct. These detection schemes are especially useful for the detectionof nucleic acid molecules if such molecules are present in very lownumbers.

In a preferred embodiment of the subject assay, the allele of an IL-1proinflammatory haplotype is identified by alterations in restrictionenzyme cleavage patterns. For example, sample and control DNA isisolated, amplified (optionally), digested with one or more restrictionendonucleases, and fragment length sizes are determined by gelelectrophoresis.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the allele. Exemplarysequencing reactions include those based on techniques developed byMaxim and Gilbert ((1977) Proc. Natl. Acad Sci USA 74:560) or Sanger(Sanger et al (1977) Proc. Nat. Acad. Sci. USA 74:5463). It is alsocontemplated that any of a variety of automated sequencing proceduresmay be utilized when performing the subject assays (see, for exampleBiotechniques (1995) 19:448), including sequencing by mass spectrometry(see, for example PCT publication WO 94/16101; Cohen et al. (1996) AdvChromatogr 36:127-162; and Griffin et al. (1993) Appl Biochem Biotechnol38:147-159). It will be evident to one of skill in the art that, forcertain embodiments, the occurrence of only one, two or three of thenucleic acid bases need be determined in the sequencing reaction. Forinstance, A-track or the like, e.g., where only one nucleic acid isdetected, can be carried out.

In a further embodiment, protection from cleavage agents (such as anuclease, hydroxylamine or osmium tetroxide and with piperidine) can beused to detect mismatched bases in RNA/RNA or RNA/DNA or DNA/DNAheteroduplexes (Myers, et al. (1985) Science 230:1242). In general, theart technique of “mismatch cleavage” starts by providing heteroduplexesformed by hybridizing (labeled) RNA or DNA containing the wild-typeallele with the sample. The double-stranded duplexes are treated with anagent which cleaves single-stranded regions of the duplex such as whichwill exist due to base pair mismatches between the control and samplestrands. For instance, RNA/DNA duplexes can be treated with RNase andDNA/DNA hybrids treated with S1 nuclease to enzymatically digest themismatched regions. In other embodiments, either DNA/DNA or RNA/DNAduplexes can be treated with hydroxylamine or osmium tetroxide and withpiperidine in order to digest mismatched regions. After digestion of themismatched regions, the resulting material is then separated by size ondenaturing polyacrylamide gels to determine the site of mutation. See,for example, Cotton et al (1988) Proc. Natl. Acad Sci USA 85:4397; andSaleeba et al (1992) Methods Enzymol. 217:286-295. In a preferredembodiment, the control DNA or RNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes). For example, the mutYenzyme of E. coli cleaves A at G/A mismatches and the thymidine DNAglycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al.(1994) Carcinogenesis 15:1657-1662). According to an exemplaryembodiment, a probe based on an allele of an IL-1 locus haplotype ishybridized to a cDNA or other DNA product from a test cell(s). Theduplex is treated with a DNA mismatch repair enzyme, and the cleavageproducts, if any, can be detected from electrophoresis protocols or thelike. See, for example, U.S. Pat. No. 5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify an IL-1 locus allele. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc Natl. Acad. Sci. USA 86:2766, see also Cotton(1993) Mutat Res 285:125-144; and Hayashi (1992) Genet Anal Tech Appl9:73-79). Single-stranded DNA fragments of sample and control IL-1 locusalleles are denatured and allowed to renature. The secondary structureof single-stranded nucleic acids varies according to sequence, theresulting alteration in electrophoretic mobility enables the detectionof even a single base change. The DNA fragments may be labeled ordetected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).

In yet another embodiment, the movement of alleles in polyacrylamidegels containing a gradient of denaturant is assayed using denaturinggradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature313:495). When DGGE is used as the method of analysis, DNA will bemodified to insure that it does not completely denature, for example byadding a GC clamp of approximately 40 bp of high-melting GC-rich DNA byPCR. In a further embodiment, a temperature gradient is used in place ofa denaturing agent gradient to identify differences in the mobility ofcontrol and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem265:12753).

Examples of other techniques for detecting alleles include, but are notlimited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonticleotide primers may be prepared in which the known mutation ornucleotide difference (e.g., in allelic variants) is placed centrallyand then hybridized to target DNA under conditions which permithybridization only if a perfect match is found (Saiki et al. (1986)Nature 324:163); Saiki et al (1989) Proc. Natl. Acad. Sci. USA 86:6230).Such allele specific oligonucleotide hybridization techniques may beused to test one mutation or polymorphic region per reaction whenoligonucleotides are hybridized to PCR amplified target DNA or a numberof different mutations or polymorphic regions when the oligonucleotidesare attached to the hybridizing membrane and hybridized with labelledtarget DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation or polymorphic region of interest in the centerof the molecule (so that amplification depends on differentialhybridization) (Gibbs et al (1989), Nucleic Acids Res. 17:2437-2448) orat the extreme 3′ end of one primer where, under appropriate conditions,mismatch can prevent, or reduce polymerase extension (Prossner (1993)Tibtech 11:238. In addition it may be desirable to introduce a novelrestriction site in the region of the mutation to create cleavage-baseddetection (Gasparini et al (1992) Mol. Cell. Probes 6:1). It isanticipated that in certain embodiments amplification may also beperformed using Taq ligase for amplification (Barany (1991) Proc. Natl.Acad. Sci. USA 88:189). In such cases, ligation will occur only if thereis a perfect match at the 3′ end of the 5′ sequence making it possibleto detect the presence of a known mutation at a specific site by lookingfor the presence or absence of amplification.

In another embodiment, identification of the allelic variant is carriedout using an oligonucleotide ligation assay (OLA), as described, e.g.,in U.S. Pat. No. 4,998,617 and in Landegren, U. et al. ((1988) Science241:1077-1080). The OLA protocol uses two oligonucleotides which aredesigned to be capable of hybridizing to abutting sequences of a singlestrand of a target. One of the oligonucleotides is linked to aseparation marker, e.g., biotinylated, and the other is detectablylabeled. If the precise complementary sequence is found in a targetmolecule, the oligonucleotides will hybridize such that their terminiabut, and create a ligation substrate. Ligation then permits the labeledoligonucleotide to be recovered using avidin, or another biotin ligand.Nickerson, D. A. et al. have described a nucleic acid detection assaythat combines attributes of PCR and OLA (Nickerson, D. A. et al. (1990)Proc. Natl. Acad. Sci. USA 87:8923-27). In this method, PCR is used toachieve the exponential amplification of target DNA, which is thendetected using OLA.

Several techniques based on this OLA method have been developed and canbe used to detect alleles of an IL-1 locus haplotype. For example, U.S.Pat. No. 5,593,826 discloses an OLA using an oligonucleotide having3′-amino group and a 5′-phosphorylated oligonucleotide to form aconjugate having a phosphoramidate linkage. In another variation of OLAdescribed in To be et al. ((1996) Nucleic Acids Res 24: 3728), OLAcombined with PCR permits typing of two alleles in a single microtiterwell. By marking each of the allele-specific primers with a uniquehapten, i.e. digoxigenin and fluorescein, each OLA reaction can bedetected by using hapten specific antibodies that are labeled withdifferent enzyme reporters, alkaline phosphatase or horseradishperoxidase. This system permits the detection of the two alleles using ahigh throughput format that leads to the production of two differentcolors.

Another embodiment of the invention is directed to kits for detecting apredisposition for developing a restenosis. This kit may contain one ormore oligonucleotides, including 5′ and 3′ oligonucleotides thathybridize 5′ and 3′ to at least one allele of an IL-1 locus haplotype.PCR amplification oligonucleotides should hybridize between 25 and 2500base pairs apart, preferably between about 100 and about 500 basesapart, in order to produce a PCR product of convenient size forsubsequent analysis.

Particularly preferred primers for use in the diagnostic method of theinvention include the following:

(SEQ ID No. 1) 5′ ATG GTT TTA GAA ATC ATC AAG CCT AGG GCA 3′ and (SEQ IDNo. 2) 5′ AAT GAA AGG AGG GGA GGA TGA CAG AAA TGT 3′ (SEQ ID No. 3)5′ TGG CAT TGA TCT GGT TCA TC-3′ and (SEQ ID No. 4) 5′ GTT TAG GAA TCTTCC CAC TT-3′; (SEQ ID No. 5) 5′ CTC AGG TGT CCT CGA AGA AAT CAA A 3′and (SEQ ID No. 6) 5′ GCT TTT TTG CTG TGA GTC CCG 3′; (SEQ ID NO. 7)5′-CTC.AGC.AAC.ACT.CCT.AT-3′ and (SEQ ID NO. 8)5′-TCC.TGG.TCT.GCA.GCT.AA-3′; (SEQ ID NO. 9) 5′-CTA TCT GAG GAA CAA ACTAGT AGC-3′ and (SEQ ID NO. 10) 5′-TAG GAC ATT GCA CCT AGG GTT TGT -3′;(SEQ. ID No. 11) 5′ ATT TTT TTA TAA ATC ATC AAG CCT AGG GCA 3′ and (SEQ.ID No. 12) 5′ AAT TAA AGG AGG GAA GAA TGA CAG AAA TGT 3′ (SEQ ID NO. 13)5′-AAG CTT GTT CTA CCA CCT GAA CTA GGC.-3′ and (SEQ ID NO. 14) 5′-TTACAT ATG AGC CTT CCA TG.-3′;

The design of additional oligonucleotides for use in the amplificationand detection of IL-1 polymorphic alleles by the method of the inventionis facilitated by the availability of both updated sequence informationfrom human chromosome 2q13—which contains the human IL-1 locus, andupdated human polymorphism information available for this locus. Forexample, the DNA sequence for the IL-1A, IL-1B and IL-1RN is shown inFIGS. 1 (GenBank Accession No. X03833), 2 (GenBank Accession No. X04500)and 3 (GenBank Accession No. X64532) respectively. Suitable primers forthe detection of a human polymorphism in these genes can be readilydesigned using this sequence information and standard techniques knownin the art for the design and optimization of primers sequences. Optimaldesign of such primer sequences can be achieved, for example, by the useof commercially available primer selection programs such as Primer 2.1,Primer 3 or GeneFisher (See also, Nicklin M. H. J., Weith A. Duff G. W.,“A Physical Map of the Region Encompassing the Human Interleukin-1α,interleukin-1β, and Interleukin-1 Receptor Antagonist Genes” Genomics19: 382 (1995); Nothwang H. G., et al. “Molecular Cloning of theInterleukin-1 gene Cluster: Construction of an Integrated YAC/PAC Contigand a partial transcriptional Map in the Region of Chromosome 2q13”Genomics 41: 370 (1997); Clark, et al. (1986) Nucl. Acids. Res.,14:7897-7914 [published erratum appears in Nucleic Acids Res., 15:868(1987) and the Genome Database (GDB) project at the URLhttp://www.gdb.org).

For use in a kit, oligonucleotides may be any of a variety of naturaland/or synthetic compositions such as synthetic oligonucleotides,restriction fragments, cDNAs, synthetic peptide nucleic acids (PNAs),and the like. The assay kit and method may also employ labeledoligonucleotides to allow ease of identification in the assays. Examplesof labels which may be employed include radio-labels, enzymes,fluorescent compounds, streptavidin, avidin, biotin, magnetic moieties,metal binding moieties, antigen or antibody moieties, and the like.

The kit may, optionally, also include DNA sampling means. DNA samplingmeans are well known to one of skill in the art and can include, but notbe limited to substrates, such as filter papers, the AmpliCard™(University of Sheffield, Sheffield, England S10 2JF; Tarlow, J W, etal., J. of Invest. Dernatol. 103:387-389 (1994)) and the like; DNApurification reagents such as Nucleon™ kits, lysis buffers, proteinasesolutions and the like; PCR reagents, such as 10× reaction buffers,thermostable polymerase, dNTPs, and the like; and allele detection meanssuch as the HinfI restriction enzyme, allele specific oligonucleotides,degenerate oligonucleotide primers for nested PCR from dried blood.

4.2.3. Pharmacogenomics

Knowledge of the particular alleles associated with restenosis, alone orin conjunction with information on other genetic defects contributing torestenosis, such as the PL(A1/A2) polymorphism in a plateletglycoprotein (See Abbate et al. (1998) Am J Cardiol 82: 524-5), allows acustomization of the restenosis therapy to the individual's geneticprofile, the goal of “pharmacogenomics”. For example, subjects having anallele 2 of any of the following markers: IL-1A (+4845), IL-1B (−511),IL-1B (+3954) or IL-1RN (VNTR) or any nucleic acid sequence in linkagedisequilibrium with any of these alleles may have or be predisposed todeveloping restenosis and may respond better to particular therapeuticsthat address the particular molecular basis of the disease in thesubject. Thus, comparison of an individual's IL-1 profile to thepopulation profile for restenosis, permits the selection or design ofdrugs or other therapeutic regimens that are expected to be safe andefficacious for a particular patient or patient population (i.e., agroup of patients having the same genetic alteration).

In addition, the ability to target populations expected to show thehighest clinical benefit, based on genetic profile can enable: 1) therepositioning of marketed drugs with disappointing market results; 2)the rescue of drug candidates whose clinical development has beendiscontinued as a result of safety or efficacy limitations, which arepatient subgroup-specific; and 3) an accelerated and less costlydevelopment for drug candidates and more optimal drug labeling (e.g.since measuring the effect of various doses of an agent on a restenosiscausative mutation is useful for optimizing effective dose).

The treatment of an individual with a particular therapeutic can bemonitored by determining protein (e.g. IL-1α, IL-1β, or IL-1Ra), mRNAand/or transcriptional level. Depending on the level detected, thetherapeutic regimen can then be maintained or adjusted (increased ordecreased in dose). In a preferred embodiment, the effectiveness oftreating a subject with an agent comprises the steps of: (i) obtaining apreadministration sample from a subject prior to administration of theagent; (ii) detecting the level or amount of a protein, mRNA or genomicDNA in the preadministration sample; (iii) obtaining one or morepost-administration samples from the subject; (iv) detecting the levelof expression or activity of the protein, mRNA or genomic DNA in thepost-administration sample; (v) comparing the level of expression oractivity of the protein, mRNA or genomic DNA in the preadministrationsample with the corresponding protein, mRNA or genomic DNA in thepostadministration sample, respectively; and (vi) altering theadministration of the agent to the subject accordingly.

Cells of a subject may also be obtained before and after administrationof a therapeutic to detect the level of expression of genes other thanan IL-1 gene to verify that the therapeutic does not increase ordecrease the expression of genes which could be deleterious. This can bedone, e.g., by using the method of transcriptional profiling. Thus, mRNAfrom cells exposed in vivo to a therapeutic and mRNA from the same typeof cells that were not exposed to the therapeutic could be reversetranscribed and hybridized to a chip containing DNA from numerous genes,to thereby compare the expression of genes in cells treated and nottreated with the therapeutic.

4.3 Restenosis Therapeutics

Modulators of IL-1 (e.g. IL-1α, IL-1β or IL-1 receptor antagonist) or aprotein encoded by a gene that is in linkage disequilibrium with an IL-1gene can comprise any type of compound, including a protein, peptide,peptidomimetic, small molecule, or nucleic acid. Preferred agonistsinclude nucleic acids (e.g. encoding an IL-1 protein or a gene that isup- or down-regulated by an IL-1 protein), proteins (e.g. IL-1 proteinsor a protein that is up- or down-regulated thereby) or a small molecule(e.g. that regulates expression or binding of an IL-1 protein).Preferred antagonists, which can be identified, for example, using theassays described herein, include nucleic acids (e.g. single (antisense)or double stranded (triplex) DNA or PNA and ribozymes), protein (e.g.antibodies) and small molecules that act to suppress or inhibit IL-1transcription and/or protein activity.

4.3.1. Effective Dose

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining The LD₅₀ (the dose lethal to 50% of thepopulation) and the Ed₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissues in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

4.3.2. Formulation and Use

Compositions for use in accordance with the present invention may beformulated in a conventional manner using one or more physiologicallyacceptable carriers or excipients. Thus, the compounds and theirphysiologically acceptable salts and solvates may be formulated foradministration by, for example, injection, inhalation or insufflation(either through the mouth or the nose) or oral, buccal, parenteral orrectal administration.

For such therapy, the compounds of the invention can be formulated for avariety of loads of administration, including systemic and topical orlocalized administration. Techniques and formulations generally may befound in Remmington's Pharmaceutical Sciences, Meade Publishing Co.,Easton, Pa. For systemic administration, injection is preferred,including intramuscular, intravenous, intraperitoneal, and subcutaneous.For injection, the compounds of the invention can be formulated inliquid solutions, preferably in physiologically compatible buffers suchas Hank's solution or Ringer's solution. In addition, the compounds maybe formulated in solid form and redissolved or suspended immediatelyprior to use. Lyophilized forms are also included.

For oral administration, the compositions may take the form of, forexample, tablets or capsules prepared by conventional means withpharmaceutically acceptable excipients such as binding agents (e.g.,pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose orcalcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talcor silica); disintegrants (e.g., potato starch or sodium starchglycolate); or wetting agents (e.g., sodium lauryl sulfate). The tabletsmay be coated by methods well known in the art. Liquid preparations fororal administration may take the form of, for example, solutions, syrupsor suspensions, or they may be presented as a dry product forconstitution with water or other suitable vehicle before use. Suchliquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound. For buccal administration thecompositions may take the form of tablets or lozenges formulated inconventional manner. For administration by inhalation, the compounds foruse according to the present invention are conveniently delivered in theform of an aerosol spray presentation from pressurized packs or anebuliser, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulating agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt. Other suitable delivery systems includemicrospheres which offer the possibility of local noninvasive deliveryof drugs over an extended period of time. This technology utilizesmicrospheres of precapillary size which can be injected via a coronarycatheter into any selected part of the e.g. heart or other organswithout causing inflammation or ischemia. The administered therapeuticis slowly released from these microspheres and taken up by surroundingtissue cells (e.g. endothelial cells).

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration bile salts and fusidic acidderivatives. In addition, detergents may be used to facilitatepermeation. Transmucosal administration may be through nasal sprays orusing suppositories. For topical administration, the oligomers of theinvention are formulated into ointments, salves, gels, or creams asgenerally known in the art. A wash solution can be used locally to treatan injury or inflammation to accelerate healing.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

4.4 Assays to Identify Restenosis Therapeutics

Based on the identification of mutations that cause or contribute to thedevelopment of restenosis, the invention further features cell-based orcell free assays, e.g., for identifying restenosis therapeutics. In oneembodiment, a cell expressing an IL-1 receptor, or a receptor for aprotein that is encoded by a gene which is in linkage disequilibriumwith an IL-1 gene, on the outer surface of its cellular membrane isincubated in the presence of a test compound alone or in the presence ofa test compound and another protein and the interaction between the testcompound and the receptor or between the protein (preferably a taggedprotein) and the receptor is detected, e.g., by using a microphysiometer(McConnell et al. (1992) Science 257:1906). An interaction between thereceptor and either the test compound or the protein is detected by themicrophysiometer as a change in the acidification of the medium. Thisassay system thus provides a means of identifying molecular antagonistswhich, for example, function by interfering with protein-receptorinteractions, as well as molecular agonist which, for example, functionby activating a receptor.

Cellular or cell-free assays can also be used to identify compoundswhich modulate expression of an IL-1 gene or a gene in linkagedisequilibrium therewith, modulate translation of an mRNA, or whichmodulate the stability of an mRNA or protein. Accordingly, in oneembodiment, a cell which is capable of producing an IL-1, or otherprotein is incubated with a test compound and the amount of proteinproduced in the cell medium is measured and compared to that producedfrom a cell which has not been contacted with the test compound. Thespecificity of the compound vis a vis the protein can be confirmed byvarious control analysis, e.g., measuring the expression of one or morecontrol genes. In particular, this assay can be used to determine theefficacy of antisense, ribozyme and triplex compounds.

Cell-free assays can also be used to identify compounds which arecapable of interacting with a protein, to thereby modify the activity ofthe protein. Such a compound can, e.g., modify the structure of aprotein thereby effecting its ability to bind to a receptor. In apreferred embodiment, cell-free assays for identifying such compoundsconsist essentially in a reaction mixture containing a protein and atest compound or a library of test compounds in the presence or absenceof a binding partner. A test compound can be, e.g., a derivative of abinding partner, e.g., a biologically inactive target peptide, or asmall molecule.

Accordingly, one exemplary screening assay of the present inventionincludes the steps of contacting a protein or functional fragmentthereof with a test compound or library of test compounds and detectingthe formation of complexes. For detection purposes, the molecule can belabeled with a specific marker and the test compound or library of testcompounds labeled with a different marker. Interaction of a testcompound with a protein or fragment thereof can then be detected bydetermining the level of the two labels after an incubation step and awashing step. The presence of two labels after the washing step isindicative of an interaction.

An interaction between molecules can also be identified by usingreal-time BIA (Biomolecular Interaction Analysis, Pharmacia BiosensorAB) which detects surface plasmon resonance (SPR), an opticalphenomenon. Detection depends on changes in the mass concentration ofmacromolecules at the biospecific interface, and does not require anylabeling of interactants. In one embodiment, a library of test compoundscan be immobilized on a sensor surface, e.g., which forms one wall of amicro-flow cell. A solution containing the protein or functionalfragment thereof is then flown continuously over the sensor surface. Achange in the resonance angle as shown on a signal recording, indicatesthat an interaction has occurred. This technique is further described,e.g., in BIAtechnology Handbook by Pharmacia.

Another exemplary screening assay of the present invention includes thesteps of (a) forming a reaction mixture including: (i) an IL-1 or otherprotein, (ii) an appropriate receptor, and (iii) a test compound; and(b) detecting interaction of the protein and receptor. A statisticallysignificant change (potentiation or inhibition) in the interaction ofthe protein and receptor in the presence of the test compound, relativeto the interaction in the absence of the test compound, indicates apotential antagonist (inhibitor). The compounds of this assay can becontacted simultaneously. Alternatively, a protein can first becontacted with a test compound for an appropriate amount of time,following which the receptor is added to the reaction mixture. Theefficacy of the compound can be assessed by generating dose responsecurves from data obtained using various concentrations of the testcompound. Moreover, a control assay can also be performed to provide abaseline for comparison.

Complex formation between a protein and receptor may be detected by avariety of techniques. Modulation of the formation of complexes can bequantitated using, for example, detectably labeled proteins such asradiolabeled, fluorescently labeled, or enzymatically labeled proteinsor receptors, by immunoassay, or by chromatographic detection.

Typically, it will be desirable to immobilize either the protein or thereceptor to facilitate separation of complexes from uncomplexed forms ofone or both of the proteins, as well as to accommodate automation of theassay. Binding of protein and receptor can be accomplished in any vesselsuitable for containing the reactants. Examples include microtitreplates, test tubes, and micro-centrifuge tubes. In one embodiment, afusion protein can be provided which adds a domain that allows theprotein to be bound to a matrix. For example, glutathione-S-transferasefusion proteins can be adsorbed onto glutathione sepharose beads (SigmaChemical, St. Louis, Mo.) or glutathione derivatized microtitre plates,which are then combined with the receptor, e.g. an ³⁵S-labeled receptor,and the test compound, and the mixture incubated under conditionsconducive to complex formation, e.g. at physiological conditions forsalt and pH, though slightly more stringent conditions may be desired.Following incubation, the beads are washed to remove any unbound label,and the matrix immobilized and radiolabel determined directly (e.g.beads placed in scintillant), or in the supernatant after the complexesare subsequently dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level ofprotein or receptor found in the bead fraction quantitated from the gelusing standard electrophoretic techniques such as described in theappended examples. Other techniques for immobilizing proteins onmatrices are also available for use in the subject assay. For instance,either protein or receptor can be immobilized utilizing conjugation ofbiotin and streptavidin. Transgenic animals can also be made to identifyagonists and antagonists or to confirm the safety and efficacy of acandidate therapeutic. Transgenic animals of the invention can includenon-human animals containing a restenosis causative mutation under thecontrol of an appropriate endogenous promoter or under the control of aheterologous promoter.

The transgenic animals can also be animals containing a transgene, suchas reporter gene, under the control of an appropriate promoter orfragment thereof. These animals are useful, e.g., for identifying drugsthat modulate production of an IL-1 protein, such as by modulating geneexpression. Methods for obtaining transgenic non-human animals are wellknown in the art. In preferred embodiments, the expression of therestenosis causative mutation is restricted to specific subsets ofcells, tissues or developmental stages utilizing, for example,cis-acting sequences that control expression in the desired pattern. Inthe present invention, such mosaic expression of a protein can beessential for many forms of lineage analysis and can additionallyprovide a means to assess the effects of, for example, expression levelwhich might grossly alter development in small patches of tissue withinan otherwise normal embryo. Toward this end, tissue-specific regulatorysequences and conditional regulatory sequences can be used to controlexpression of the mutation in certain spatial patterns. Moreover,temporal patterns of expression can be provided by, for example,conditional recombination systems or prokaryotic transcriptionalregulatory sequences. Genetic techniques, which allow for the expressionof a mutation can be regulated via site-specific genetic manipulation invivo, are known to those skilled in the art.

The transgenic animals of the present invention all include within aplurality of their cells a restenosis causative mutation transgene ofthe present invention, which transgene alters the phenotype of the “hostcell”. In an illustrative embodiment, either the cre/loxP recombinasesystem of bacteriophage P1 (Lakso et al. (1992) PNAS 89:6232-6236; Orbanet al. (1992) PNAS 89:6861-6865) or the FLP recombinase system ofSaccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355;PCT publication WO 92/15694) can be used to generate in vivosite-specific genetic recombination systems. Cre recombinase catalyzesthe site-specific recombination of an intervening target sequencelocated between loxP sequences. loxP sequences are 34 base pairnucleotide repeat sequences to which the Cre recombinase binds and arerequired for Cre recombinase mediated genetic recombination. Theorientation of loxP sequences determines whether the intervening targetsequence is excised or inverted when Cre recombinase is present(Abremski et al. (1984) J. Biol. Chem. 259:1509-1514); catalyzing theexcision of the target sequence when the loxP sequences are oriented asdirect repeats and catalyzes inversion of the target sequence when loxPsequences are oriented as inverted repeats.

Accordingly, genetic recombination of the target sequence is dependenton expression of the Cre recombinase. Expression of the recombinase canbe regulated by promoter elements which are subject to regulatorycontrol, e.g., tissue-specific, developmental stage-specific, inducibleor repressible by externally added agents. This regulated control willresult in genetic recombination of the target sequence only in cellswhere recombinase expression is mediated by the promoter element. Thus,the activation of expression of the causative mutation transgene can beregulated via control of recombinase expression.

Use of the cre/loxP recombinase system to regulate expression of acausative mutation transgene requires the construction of a transgenicanimal containing transgenes encoding both the Cre recombinase and thesubject protein. Animals containing both the Cre recombinase and therestenosis causative mutation transgene can be provided through theconstruction of “double” transgenic animals. A convenient method forproviding such animals is to mate two transgenic animals each containinga transgene.

Similar conditional transgenes can be provided using prokaryoticpromoter sequences which require prokaryotic proteins to be simultaneousexpressed in order to facilitate expression of the transgene. Exemplarypromoters and the corresponding trans-activating prokaryotic proteinsare given in U.S. Pat. No. 4,833,080.

Moreover, expression of the conditional transgenes can be induced bygene therapy-like methods wherein a gene encoding the transactivatingprotein, e.g. a recombinase or a prokaryotic protein, is delivered tothe tissue and caused to be expressed, such as in a cell-type specificmanner. By this method, the transgene could remain silent into adulthooduntil “turned on” by the introduction of the transactivator.

In an exemplary embodiment, the “transgenic non-human animals” of theinvention are produced by introducing transgenes into the germline ofthe non-human animal. Embryonal target cells at various developmentalstages can be used to introduce transgenes. Different methods are useddepending on the stage of development of the embryonal target cell. Thespecific line(s) of any animal used to practice this invention areselected for general good health, good embryo yields, good pronuclearvisibility in the embryo, and good reproductive fitness. In addition,the haplotype is a significant factor. For example, when transgenic miceare to be produced, strains such as C57BL/6 or FVB lines are often used(Jackson Laboratory, Bar Harbor, Me.). Preferred strains are those withH-2^(b), H-2^(d) or H-2^(q) haplotypes such as C57BL/6 or DBA/1 Theline(s) used to practice this invention may themselves be transgenics,and/or may be knockouts (i.e., obtained from animals which have one ormore genes partially or completely suppressed).

In one embodiment, the transgene construct is introduced into a singlestage embryo. The zygote is the best target for microinjection. In themouse, the male pronucleus reaches the size of approximately 20micrometers in diameter which allows reproducible injection of 1-2 μl ofDNA solution. The use of zygotes as a target for gene transfer has amajor advantage in that in most cases the injected DNA will beincorporated into the host gene before the first cleavage (Brinster etal. (1985) PNAS 82:4438-4442). As a consequence, all cells of thetransgenic animal will carry the incorporated transgene. This will ingeneral also be reflected in the efficient transmission of the transgeneto offspring of the founder since 50% of the germ cells will harbor thetransgene.

Normally, fertilized embryos are incubated in suitable media until thepronuclei appear. At about this time, the nucleotide sequence comprisingthe transgene is introduced into the female or male pronucleus asdescribed below. In some species such as mice, the male pronucleus ispreferred. It is most preferred that the exogenous genetic material beadded to the male DNA complement of the zygote prior to its beingprocessed by the ovum nucleus or the zygote female pronucleus. It isthought that the ovum nucleus or female pronucleus release moleculeswhich affect the male DNA complement, perhaps by replacing theprotamines of the male DNA with histones, thereby facilitating thecombination of the female and male DNA complements to form the diploidzygote. Thus, it is preferred that the exogenous genetic material beadded to the male complement of DNA or any other complement of DNA priorto its being affected by the female pronucleus. For example, theexogenous genetic material is added to the early male pronucleus, assoon as possible after the formation of the male pronucleus, which iswhen the male and female pronuclei are well separated and both arelocated close to the cell membrane. Alternatively, the exogenous geneticmaterial could be added to the nucleus of the sperm after it has beeninduced to undergo decondensation. Sperm containing the exogenousgenetic material can then be added to the ovum or the decondensed spermcould be added to the ovum with the transgene constructs being added assoon as possible thereafter.

Introduction of the transgene nucleotide sequence into the embryo may beaccomplished by any means known in the art such as, for example,microinjection, electroporation, or lipofection. Following introductionof the transgene nucleotide sequence into the embryo, the embryo may beincubated in vitro for varying amounts of time, or reimplanted into thesurrogate host, or both. In vitro incubation to maturity is within thescope of this invention. One common method in to incubate the embryos invitro for about 1-7 days, depending on the species, and then reimplantthem into the surrogate host.

For the purposes of this invention a zygote is essentially the formationof a diploid cell which is capable of developing into a completeorganism. Generally, the zygote will be comprised of an egg containing anucleus formed, either naturally or artificially, by the fusion of twohaploid nuclei from a gamete or gametes. Thus, the gamete nuclei must beones which are naturally compatible, i.e., ones which result in a viablezygote capable of undergoing differentiation and developing into afunctioning organism. Generally, a euploid zygote is preferred. If ananeuploid zygote is obtained, then the number of chromosomes should notvary by more than one with respect to the euploid number of the organismfrom which either gamete originated.

In addition to similar biological considerations, physical ones alsogovern the amount (e.g., volume) of exogenous genetic material which canbe added to the nucleus of the zygote or to the genetic material whichforms a part of the zygote nucleus. If no genetic material is removed,then the amount of exogenous genetic material which can be added islimited by the amount which will be absorbed without being physicallydisruptive. Generally, the volume of exogenous genetic material insertedwill not exceed about 10 picoliters. The physical effects of additionmust not be so great as to physically destroy the viability of thezygote. The biological limit of the number and variety of DNA sequenceswill vary depending upon the particular zygote and functions of theexogenous genetic material and will be readily apparent to one skilledin the art, because the genetic material, including the exogenousgenetic material, of the resulting zygote must be biologically capableof initiating and maintaining the differentiation and development of thezygote into a functional organism.

The number of copies of the transgene constructs which are added to thezygote is dependent upon the total amount of exogenous genetic materialadded and will be the amount which enables the genetic transformation tooccur. Theoretically only one copy is required; however, generally,numerous copies are utilized, for example, 1,000-20,000 copies of thetransgene construct, in order to insure that one copy is functional. Asregards the present invention, there will often be an advantage tohaving more than one functioning copy of each of the inserted exogenousDNA sequences to enhance the phenotypic expression of the exogenous DNAsequences.

Any technique which allows for the addition of the exogenous geneticmaterial into nucleic genetic material can be utilized so long as it isnot destructive to the cell, nuclear membrane or other existing cellularor genetic structures. The exogenous genetic material is preferentiallyinserted into the nucleic genetic material by microinjection.Microinjection of cells and cellular structures is known and is used inthe art.

Reimplantation is accomplished using standard methods. Usually, thesurrogate host is anesthetized, and the embryos are inserted into theoviduct. The number of embryos implanted into a particular host willvary by species, but will usually be comparable to the number of offspring the species naturally produces.

Transgenic offspring of the surrogate host may be screened for thepresence and/or expression of the transgene by any suitable method.Screening is often accomplished by Southern blot or Northern blotanalysis, using a probe that is complementary to at least a portion ofthe transgene. Western blot analysis using an antibody against theprotein encoded by the transgene may be employed as an alternative oradditional method for screening for the presence of the transgeneproduct. Typically, DNA is prepared from tail tissue and analyzed bySouthern analysis or PCR for the transgene. Alternatively, the tissuesor cells believed to express the transgene at the highest levels aretested for the presence and expression of the transgene using Southernanalysis or PCR, although any tissues or cell types may be used for thisanalysis.

Alternative or additional methods for evaluating the presence of thetransgene include, without limitation, suitable biochemical assays suchas enzyme and/or immunological assays, histological stains forparticular marker or enzyme activities, flow cytometric analysis, andthe like. Analysis of the blood may also be useful to detect thepresence of the transgene product in the blood, as well as to evaluatethe effect of the transgene on the levels of various types of bloodcells and other blood constituents.

Progeny of the transgenic animals may be obtained by mating thetransgenic animal with a suitable partner, or by in vitro fertilizationof eggs and/or sperm obtained from the transgenic animal. Where matingwith a partner is to be performed, the partner may or may not betransgenic and/or a knockout; where it is transgenic, it may contain thesame or a different transgene, or both. Alternatively, the partner maybe a parental line. Where in vitro fertilization is used, the fertilizedembryo may be implanted into a surrogate host or incubated in vitro, orboth. Using either method, the progeny may be evaluated for the presenceof the transgene using methods described above, or other appropriatemethods.

The transgenic animals produced in accordance with the present inventionwill include exogenous genetic material. Further, in such embodimentsthe sequence will be attached to a transcriptional control element,e.g., a promoter, which preferably allows the expression of thetransgene product in a specific type of cell.

Retroviral infection can also be used to introduce the transgene into anon-human animal. The developing non-human embryo can be cultured invitro to the blastocyst stage. During this time, the blastomeres can betargets for retroviral infection (Jaenich, R. (1976) PNAS 73:1260-1264).Efficient infection of the blastomeres is obtained by enzymatictreatment to remove the zona pellucida (Manipulating the Mouse Embryo,Hogan eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,1986). The viral vector system used to introduce the transgene istypically a replication-defective retrovirus carrying the transgene(Jahner et al. (1985) PNAS 82:6927-6931; Van der Putten et al. (1985)PNAS 82:6148-6152). Transfection is easily and efficiently obtained byculturing the blastomeres on a monolayer of virus-producing cells (Vander Putten, supra; Stewart et al. (1987) EMBO J. 6:383-388).Alternatively, infection can be performed at a later stage. Virus orvirus-producing cells can be injected into the blastocoele (Jahner etal. (1982) Nature 298:623-628). Most of the founders will be mosaic forthe transgene since incorporation occurs only in a subset of the cellswhich formed the transgenic non-human animal. Further, the founder maycontain various retroviral insertions of the transgene at differentpositions in the genome which generally will segregate in the offspring.In addition, it is also possible to introduce transgenes into the germline by intrauterine retroviral infection of the midgestation embryo(Jahner et al. (1982) supra).

A third type of target cell for transgene introduction is the embryonalstem cell (ES). ES cells are obtained from pre-implantation embryoscultured in vitro and fused with embryos (Evans et al. (1981) Nature292:154-156; Bradley et al. (1984) Nature 309:255-258; Gossler et al.(1986) PNAS 83: 9065-9069; and Robertson et al. (1986) Nature322:445-448). Transgenes can be efficiently introduced into the ES cellsby DNA transfection or by retrovirus-mediated transduction. Suchtransformed ES cells can thereafter be combined with blastocysts from anon-human animal. The ES cells thereafter colonize the embryo andcontribute to the germ line of the resulting chimeric animal. For reviewsee Jaenisch, R. (1988) Science 240:1468-1474.

The present invention is further illustrated by the following exampleswhich should not be construed as limiting in any way. The contents ofall cited references (including literature references, issued patents,published patent applications as cited throughout this application) arehereby expressly incorporated by reference. The practice of the presentinvention will employ, unless otherwise indicated, conventionaltechniques that are within the skill of the art. Such techniques areexplained fully in the literature. See, for example, Molecular Cloning ALaboratory Manual, (2nd ed., Sambrook, Fritsch and Maniatis, eds., ColdSpring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D.N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984);U.S. Pat. No. 4,683,195; U.S. Pat. No. 4,683,202; and Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds., 1984).

5. EXAMPLES 5.1. IL-1RN*2 Allele Association with Decreased Risk ofRestenosis

In this example, DNA samples collected form 171 patients attending forelective percutaneous transluminal coronary angioplasty were studied at4 and 6 months post-surgery using angiography. At follow-up angiography,the patients were separated into restenosers (>50% luminal narrowing)and non-restenosers (<50% luminal narrowing), and were further assessedfor their genotype at the following IL-1 polymorphisms: IL-1A (−889),IL-1B (−511), IL-1B (+3954), IL-1RN (intron 2 VNTR).

Methods

Patients

171 patients who were scheduled to undergo follow-up angiography afterelective PTCA without stenting as part of other protocols were studied.Quantitive coronary angiography was performed on-line (Philips IntegrisHM 3000, (S); Siemens Micor (L)). Patients were electively recruited inSheffield where follow-up angiography was performed at 6 months. 117patients were recruited from Leicester. These patients had been part ofthe SHARP study (subcutaneous Heparin and Angioplasty RestenosisPrevention) where follow-up had been performed at 4 months ±2 weeks(Samani N J, et al., Lancet. 1995; 345:1013-1016), and 67% of theoriginal cohort were electively recalled for the current study. TheSHARP study did not show any effect of subcutaneous heparin upon ratesof restenosis.

A dichotomous definition of restenosis was used setting restenosis as aluminal narrowing >50% and non-restenosis <50%, at follow-upangiography. Using this definition, the cohort comprised 39% restenosersand 61% non-restenosers.

These studies were approved by the North Sheffield Ethics Committee andby the Leicester Ethics Committee, and patients gavie their writteninformed consent.

Analysis of Genetic Polymorphisms

Genomic DNA was extracted from whole blood using standard methods andPCR for variants within the IL-1 locus performed as previously described(Francis S E, et al. Circulation 1999; 99:861-866). or using anautomated Taqman™ FRET-based system. The less common IL-RN gene variantis referred to as IL-1RN.*2.

Differences in genotype distribution were assessed by chi-squareanalysis of the relevant 2*2 contingency table (table 2). Odds ratioswith 95% confidence intervals were also calculated. To summarize resultsover the Leicester and Sheffield cohorts Mantel Haenszel analyses wereperformed. A p-value of less than 0.05 was used to indicate nominalsignificance. For an overall type I error of 0.05, a corrected criticalp-value of 0.013 should be used to account for multiple testing. Here wehave corrected accounting for the 4 loci tested. However, due to linkagedisequilibrium between these loci, this correction is likely to beconservative. IL-1RN (VNTR) was collapsed and analysed as a biallelicmarker since very few genotypes were recorded with the rarer alleles.Neither of the cohorts studied were significantly different from theHardy Weinberg Equilibrium for any of the polymorphisms.

Demographic data were expressed as percent with actual counts inparentheses. These variables were compared by χ² test.

Results

Demographics

The Sheffield and Leicester combined cohorts were well matched forbaseline clinical features (Table 1).

Genetic Analysis

The Mantel-Haenzel results summarized over the Leicester and Sheffieldcohorts showed no significant differences in genotypic distributions atthe IL-1A (−889), IL-1B (+3954) and IL-1B (−511) loci betweenrestenosers and non-restenosers (Table II).

The frequency of allele 2 (IL-1RN*2) was however increased in thenon-restenosers: 34% versus 23% in restenosers (FIG. 1, Table II).Genotype distribution analysis indicated a significant associationbetween homozygosity for allele*2 and non-restenosis (MH, p=0.0196(L+S); p=0.0131 (L+S, SVD only, Table III)). When the populations areanalysed separately, the data trends concur, but are only significant inthe Sheffield SVD cohort (p=0.0384 (S); p=0.1573 (L)). This is mostlikely because of the low statistical power of these tests, since samplesizes are small due to data subdivision.

Interestingly, and a further implication that the results are morespecifically applicable to SVD only, when carriage of IL-1RN*2 iscompared between SVD and MVD groups in the Leicester cohort, there is asignificant increase of carriage of IL-RN*2 in the SVD group (p=0.0342).This result is strengthened when the Sheffield SVD patients are added(p=0.0314).

Discussion

These data suggest a genetic susceptibility to restenosis mediated bypolymorphism at the IL-1 locus.

Specifically, the data presented here indicate that IL-1RN*2 isassociated with a lower restenosis rate in patients with SVD. Thissupports previous data indicating that distinct populations withdifferent prepensitites to restenosis exist, and that the precess is atleast to some extent patient-related rather than lesion dependent orboth (Lehmann K G, et al. Circulation, 1996; 93:1123-1132; Weintraub WS, et al. Am J. Cardiol. 1993; 72:1107-1113). Our previous data (FrancisS E, et al. Circulation 1999; 99:861-866), that IL-1RN*2 is associatedwith SVD on the basis of angiography, led us to speculate that there maybe a true genetic distinction between SVD and MVD. If so, this mightindicate that IL-1RN*2 genotype could either lead more rapidly to SVD orprotest against progression to MVD. The data presented here add to this.

Since restenosis is a biological phenomenon characterized by an earlyinflammatory response, these new data suggest that IL-1RN*2 may modulatethe arterial wall response to injury in such a way as to reduce thelikelihood of restenosis. Whilst there are many potential mechanisms bywhich this could occur, a protection or beneficial effect of IL-1RN*2upon vessel wall healing in response to injury is suggested. This mightalso support the hypothesis that IL-1RN*2 slows progression toward MVDmade in our earlier study (Francis S E, et al. Circulation 1999;99:861-866).

The mechanism by which IL-1RN*2 modulates the vessel wall response toinjury is unclear. This polymorphism has functional correlates but theseappear highly cell-type specific. In monocytes, IL-1RN*2 is associatedwith increased IL-1ra production under basal and stimulated conditions(Wilkinson R J, et al. J Exp Med. 1999; 189: 1863-1873). In contrast,within cells of the columnar epithelium in inflammatory bowel disease(Carter M J, Gastroenterology. 1998; 114(4):3882), and in endothelialcells (Dewberry R M, et al. Heart. 1999; 81 (Suppl 1); 78 [abstract]),IL-1RN*2 os associated with reduced production of IL-1ra. Since theinflammatory influx seen following experimental PTCA in pigs is highlyneutrophilic and IL-1B staining abundant, predominantly in the luminalendothelium even into the late phase of healing (Chamberlin J, et al.Cardiovasc Res. 1999; 44(1):156-165), we speculate that the relativelypro-inflammatory endothelial cell phenotype created by the IL-1RN*2genotype may be important to PTCA. This suggests that modifying theinflammatory response at the time of injury may indeed be beneficialacting to limit the healing response that leads to luminal re-narrowing.

The IL-1RN VNTR polymorphism is known to be in linkage disequilibriumwith other genes in the IL-1 locus (Cox A, et al. Am J Hum Genet. 1998;62(5):1180-1188), and although there are some weakly consistent trendswhich exist for IL-1A (=4845) and IL-1B (+3954), there are no othersignificant associations with restenosis or non-restenosis for the otherIL-1 polymorphisms within the cluster. Hence, a specific complexhaplotype is not supported by these data. However, linkagedisequilibrium between this polymorphism and other unidentified genepolymorphisms cannot be excluded.

Due to sub-division of the data, this study has small sample sizes formany of the analyses performed. This reduces power and to some extentthe reliability and confidence in these findings. However, the resultshere are strengthened by the fact that two separate cohorts werecollected, and that very similar directional trends were found in bothpopulations. It was consistently found that evidence for association wasstrengthened by summarizing over the two cohorts, which furtherillustrates the concordance. It is, of course, possible that spuriousresults could have arisen due to genetic admixture within the cohorts,but again the consistence between the two populations argues away fromthis.

We favor the interpretation that polymorphic variation within the IL-1locus has an important inpact on arterial disease. Our originalpublished work (Francis S E, et al. Circulation 1999; 99:861-866).showed an association with single vessel coronary disease in twoindependent populations (Sheffield and London). The study reported hereshows association with a different clinical phenotype in a populationpredominantly from Leicester. Other investigators have demonstratedassociation between IL-1RN+2016, a single nucleotide polymorphism (SNP)in linkage disequilibrium with IL-1RN*2, and carotid intimal/medialchanges in African Americans (Pankow J S, et al. Association ofInterleukin-1 gene variants and carotid arterial wall thickness: theARID Study. 71st EAS Congress and Satellite Symposia) These all arguestrongly that polymorphism within the IL-1 locus does have an impact onthe pathogenesis of atherosclerotic lesions, although the mechanismremains to be elucidated.

The biological control of IL-1 is complex (Dinarello C A. Blood. 1991;77:1627-1632). IL-1 actions are inhibited by a non-signaling receptorIL-1 RII in membrane bound or soluble form and also by IL-1ra (Symons JA, et al. J Exp Med. 1991; 177:557-560) which binds without agonistactivity to be signaling receptor IL-1RI (Symons J A, et al. Proc NatlAcad Sci. 1995; 92:1714-1718). IL-1ra is an acute phase protein andinduced by cytokines and bacterial products (Arend W P. Adv Immunol.1993; 54:167-227). Levels of IL-1 and IL-1ra in vivo vary in parallelsuggesting a coordinated pattern of regulation (Arend WP. Adv Immunol.1993; 54:167-227). IL-1ra is detected in the endothelium of diseasedcoronary arteries (Dewberry R M, et al. Heart. 1999; 81(Suppl 1); 78[abstract]) and inhibits fatty streak formation in the apolipoprotein Edeficient mouse (Hirsch E, et al. Proc Natl Acad. Sci. 1996;93:11008-11013). These data taken together strongly implicate IL-1ra inthe control of inflammation in the arterial wall.

In conclusion, the results reported here suggest an importantassociation between IL-1RN*2 and protection from restenosis inindividuals with SVD. They also might suggest that inflammation may be apositive influence rather than wholly negative after arterial injury.Validation studies in larger study groups including a post-stenting anda reappraisal of the complex injury-repair mechanisms employed by thearterial wall are indicated.

TABLE I Clinical Characteristics of Patients with and without RestenosisRestenosis Non-restenosis P Leicester no. of patients 49 69 age (yrs)mean ± SEM 59.08 ± 1.19 57.08 ± 0.91 ns Women (%) 12.2 [6]  17.4 [12] ndHypertnesion (%)   24 [12] 17.3 [12] nd Smoking (%)   29 [14] 34.7 [24]nd Diabetes (%) 2.04 [1]  4.34 [3]  nd MI (%) 48.9 [24] 43.4 [30] ndMultivessel disease (%) 48.9 [24] 39.1 [27] nd Sheffield no. of patients18 35 age (yrs) mean ± SEM 53.61 ± 1.77 53.88 ± 1.45 ns Women (%)  17[3] 11.4 [4]  nd Hypertension (%) 61.1 [11] 37.1 [13] nd Smoking (%)77.7 [14] 74.2 [26] nd Diabetes (%) 5.5 [1] 11.4 [4]  nd MI (%) 57.1[8]  42.8 [15] nd Multivessel disease (%)  0  0 nd Sheffield andLeicester no. of patients 67 104  age (yrs) mean ± SEM 57.97 ± 1.4455.98 ± 0.98 ns Women (%) 13.4 [9]  15.3 [16] ns Hypertension (%) 34.3[23] 24.0 [25] ns Smoking (%) 41.7 [28] 48.0 [50] ns Diabetes (%) 2.98[2]  6.7 [7] ns MI (%) 47.7 [32] 43.2 [45] ns Multivessel disease (%)35.8 [24] 25.9 [27] ns Values in parentheses are the number of patientsaffected in that cohort. Hypertension defined as diastolic bp > 95 mmHg(Leicester); Sytolic bp > 160 mmHg. Smoking: current or former(Sheffield), current (Leicester). ns not significant, where normalstatistical significance, P < 0.05. nd—not done.

TABLE II Carriage of alleles within the IL-1 locus in Sheffield andLeicester restenosis and non-restenosis cohorts. 11 12/22 11 12/22 11/1222 11/12 22 Leicester SVD &MVD restenosis 20 24 26 19 36 7 46 3 non 3427 42 19 47 12 58 10 p-value 0.2399 0.2982 0.6194 0.6191 OR 1.6 1.5 1.32.6 95% CI 0.7, 3.6 0.7, 3.3 0.5, 3.7 0.7, 10.2 Sheffield SVD onlyrestenosis 8 10 10 8 15 3 16 0 non 20 14 22 11 29 4 25 7 p-value 0.43290.3244 0.6691 0.0384 OR 1.6 1.8 0.7 N/A 95% CI 0.5, 5.2 0.6, 5.7 01.,3.5 N/A MH p-value 0.1604 0.1594 0.8333 0.0196 MH Mantel-Haenzselsummary statistic N/A OR and p-value not applicable since one of thevalues in the contingency table is 0. Note: Alleles are groupedaccording to previously described commonest haplotype (Cox A, et al. AmJ Hum Genet. 1998; 62(5): 1180-1188), carriage of *2 for IL-1A [+4845];IL1B [+3954] and carriage of *1 for IL-1B [−511] and IL-1Rn [VNTR].

TABLE III Homozygosity at IL-1RN*2 illustrates the difference betweenSVD and MVD in the Sheffield and Leicester cohorts. SVD MVD 11/12 2211/12 22 Leicester restenosis 24 1 22 2 non 35 7 23 3 p-value 0.15730.7699 OR 4.8 1.4 95% CI 0.6, 41.6 0.1, 4.6 Sheffield restenosis 16 0N/A non 25 7 p-value 0.0384 OR N/A 95% CI MH p-value 0.0131 MH MantelNaenszel summary statistic N/A OR and p-value not applicable since oneof the values in the contingency table is 0.

5.2. Protective Role Against Restenosis from an Interleukin-1 ReceptorAntagonist Gene Polymorphism in Patients Treated with Coronary Stenting(The Munich Study)

Patients

The study included 1850 consecutive Caucasian patients with symptomaticcoronary artery disease who underwent coronary stent implantation atDeutsches Herzzentrurn München and 1. Medizinische Klinik rechts derIsar der Technischen Universität München. All patients were scheduledfor angiographic follow-up at 6 months. All patients participating inthis study gave written informed consent for the intervention, follow-upangiography, and genotype determination. The study protocol conformed tothe Declaration of Helsinki and was approved by the institutional ethicscommittee.

TABLE 3 Baseline clinical characteristics. IL-1RN 1/2 or 2/2 IL-1RN 1/1(n = 896) (n = 954) P Age - yr 63.4 ± 10.0 62.6 ± 10.0 0.11 Women - %22.4 19.9 0.19 Arterial hypertension - % 67.2 68.9 0.44 Diabetes - %22.7 19.4 0.08 Current or former smoker - % 38.7 41.2 0.28 Elevatedtotal cholesterol - % 42.5 43.1 0.81 Acute myocardial infarction - %20.3 20.2 0.97 Unstable angina - % 27.9 27.8 0.95 Prior bypass surgery -% 10.6 11.5 0.53 Reduced left ventricular function - % 31.3 27.7 0.09Number of diseased coronary vessels 0.39 1 vessel - % 29.2 27.3 2vessels - % 32.9 31.9 3 vessels - % 37.8 40.9 Periprocedural abciximabtherapy - % 19.8 19.6 0.93 Data are proportions or mean SDThe protocol of stent placement and poststenting therapy is familiar topractitioners in the arts. Most of the stents were implantedhand-mounted on conventional angioplasty balloons. Postproceduraltherapy consisted of aspirin (100 mg twice daily, indefinitely) andticlopidine (250 mg twice daily for 4 weeks). Patients with suboptimalresults due to residual thrombus or dissection with flow impairmentafter stent implantation received additional therapy with abciximabgiven as bolus injection during stent insertion procedure and as a12-hours continuous infusion thereafter. The decision to give abciximabwas taken at the operator's discretion.

Determination of the IL-1RN Genotype

Genomic DNA was extracted from 200 ml of peripheral blood leukocyteswith the QIAamp Blood Kit (Qiagen, Hilden, Germany) and the High PurePCR Template Preparation Kit (Boehringer Mannheim, Mannheim, Germany).

IL-1RN genotyping was performed with the ABI Prism Sequence DetectionSystem (PE Applied Biosystems, Weiterstadt, Germany). The use ofallele-specific fluorogenic probes in the 5′ nuclease reaction combinesDNA amplification and genotype determination into a single assay 33.IL-1RN (+2018), a single base pair polymorphism in exon 2, was thepolymorphism typed for this study 26. The nucleotide sequences ofprimers and probes were as follows: forward primer 5′ GGG ATG TTA ACCAGA AGA CCT TCT ATC T 3′(SEQ ID NO. 22), reverse primer 5′ CAA CCA CTCACC TTC TAA ATT GAC ATT 3′ (SEQ ID NO. 23), allele 1 probe 5′ AAC AACCAA CTA GTT GCT GGA TAC TTG CAA 3′(SEQ ID NO. 24), allele 2 probe 5′ ACAACC AAC TAG TTG CCG GAT ACT TGC 3′(SEQ ID NO. 25). The probes for allele1 were labeled with the fluorescent dye 6-carboxy-fluorescein (FAM) andfor allele 2 with the fluorescent dye tetrachloro-6-carboxy-fluorescein(TET) at the 5′ end. Both probes were labeled with the quencher6-carboxy-tetramethyl-rhodamine (TAMRA) at their 3′ ends. Thethermocycling protocol consisted of 40 cycles of denaturation at 95 Cfor 15 seconds and annealing/extension at 64 C for 1 minute. Genotypevalidation was performed by repeating the determination in 20% of thepatients using a duplicate DNA sample with a novel subject codeunrelated to the original subject code. There was a 100% matchingbetween the 2 results.

Angiographic Assessment

Coronary lesions were classified according to the modified AmericanCollege of Cardiology/American Heart Association grading system. Leftventricular function was assessed qualitatively on the basis of biplaneangiograms using a 7 segment division; the diagnosis of reduced leftventricular function was established in the presence of at least twohypokinetic segments in the contrast angiogram. Quantitativecomputer-assisted angiographic analysis was performed off-line onangiograms obtained just before stenting, immediately after stenting,and at follow up using the automated edge-detection system CMS (MedisMedical Imaging Systems, Nuenen, The Netherlands). Operators wereunaware of the patient's IL-1RN genotype. Identical projections of thetarget lesion were used for all assessed angiograms. Minimal lumendiameter, interpolated reference diameter, diameter stenosis, lesionlength and diameter of the maximally inflated balloon were theangiographic parameters obtained with this analysis system. Acute lumengain was calculated as the difference between minimal lumen diameter atthe end of intervention and minimal lumen diameter before theintervention. Late lumen loss was calculated as the difference betweenminimal lumen diameter at the end of intervention and minimal lumendiameter at the time of follow-up angiography. Loss index was calculatedas the ratio between late lumen loss and acute lumen gain.

DEFINITIONS AND STUDY ENDPOINTS

Primary endpoint of the study was restenosis. Two measures of restenosiswere assessed: the incidence of angiographic restenosis defined as adiameter stenosis of 50% at 6-month follow-up angiography, and the needfor target vessel revascularization (PTCA or aortocoronary bypasssurgery [CABG]) due to symptoms or signs of ischemia in the presence ofangiographic restenosis at the stented site over 1 year after theintervention. Other major adverse events evaluated were: death from anycause and myocardial infarction. All deaths were considered due tocardiac causes unless an autopsy established a noncardiac cause. Thediagnosis of acute myocardial infarction was based on the criteriaapplied in the EPISTENT trial (new pathological Q waves or a value ofcreatine kinase [CK] or its MB isoenzyme at least 3 times the upperlimit) 35. CK was determined systematically over the 48 hours followingstenting procedure. Clinical events were monitored throughout the 1-yearfollow-up period. The assessment was made on the basis of theinformation provided by hospital readmission records, referringphysician or phone interview with the patient. For all those patientswho revealed cardiac symptoms during the interview, at least oneclinical and electrocardiographic check-up was performed at theoutpatient clinic or by the referring physician.

Statistical Analysis

Discrete variables are expressed as counts or percentages and comparedwith Chi-square or Fisher's exact test, as appropriate. Continuousvariables are expressed as mean SD and compared by means of theunpaired, two-sided t-test or analysis of variance for more than 2groups. Risk analysis was performed calculating the odds ratio and the95% confidence interval. The main analysis consisted in comparingcombined heterozygous and homozygous carriers of the IL-11RN*2 allelewith homozygous carriers of the IL-11RN*1 allele. Moreover, theassociation between IL-1RN genotype and restenosis was assessed in amultivariate logistic regression model including also those clinical andlesion-related characteristics for which the comparison between carriersand noncarriers of the IL-1RN*2 allele showed a P-value 0.30. In thismultivariate model, we tested for the possible interaction betweenIL-1RN genotype and age. Since the relative contribution of geneticfactors to multifactorial processes such as restenosis may decrease withthe age, we carried out an additional analysis for a prespecifiedsubgroup of patients <60 years. Successively, we used test for trend forassessing gene dose effect, i.e. a stepwise increasing phenotypicresponse with the presence of 0, 1 or 2 putative alleles. Statisticalsignificance was accepted for P-values 0.05.

Results Patients Characteristics

The observed IL-1RN genotypes in the study population were 1/1 in 954(51.6%), 1/2 in 742 (40.1%) and 2/2 in 154 (8.3%). Thus, allele 2frequency was 0.28. The observed distribution complied withHardy-Weinberg equilibrium. Main baseline characteristics of thepatients are listed in Table 6 and compared between carriers andnoncarriers of the IL-1RN*2 allele. There was a trend to a higherfrequency of diabetes and reduced left ventricular function amongcarriers of the IL-1RN*2 allele. The other characteristics were evenlydistributed between the 2 groups. The angiographic and proceduralcharacteristics at the time of intervention are listed in Table 7 andshow no significant differences between carriers and noncarriers of theIL-1RN*2 allele.

TABLE 5 Lesion and procedural characteristics at the time ofintervention. IL-1RN 1/2 or 2/2 IL-1RN 1/1 (n = 896) (n = 954) P Targetcoronary vessels 0.89 Left main - % 1.3 1.6 LAD - % 40.1 39.3 LCx - %19.9 20.0 RCA - % 32.6 31.9 Venous bypass graft - % 6.1 7.2 Complexlesions - % 75.2 74.1 0.58 Restenotic lesions - % 25.3 23.3 0.30 Beforestenting Reference diameter, mm 3.02 ± 0.53 3.05 ± 0.54 0.29 Diameterstenosis - % 79.1 ± 14.9 78.7 ± 15.7 0.57 Lesion length - mm 12.1 ± 6.9 12.1 ± 6.6  0.98 Procedural data Measured balloon diameter - 3.2 ± 0.53.2 ± 5   0.45 mm Maximal balloon pressure - atm 13.9 ± 3.3  13.8 ± 3.2 0.20 Stented segment length - mm 20.0 ± 14.3 20.3 ± 13.6 0.70Immediately after stenting Diameter stenosis - % 5.2 ± 9.1 5.4 ± 7.60.47 Data are proportions or mean ± SD LAD indicates left anteriordescending coronary artery; LCx, left circumflex coronary artery; RCA,right coronary artery; complex lesions were defined as ACC/AHA lesiontypes B2 and C, according to the American College of Cardiology/AmericanHeart Association grading system.

IL-1 RN Polymorphism, Mortality and Myocardial Infarction After Stenting

Table 6 shows the adverse clinical events observed within the first 30days after coronary stenting in carriers and noncarriers of the IL-1RN*2allele. There was no association between the presence of the IL-1RN*2allele and death, myocardial infarction or target vesselrevascularization, showing no significant influence of the polymorphismin the IL-1ra gene in the risk for early thrombotic events aftercoronary stenting.

TABLE 6 Incidence of adverse events recorded during the early 30 daysIL-1RN 1/2 or 2/2 IL-1RN 1/1 (n = 896) (n = 954) P Death - % 0.9 0.90.91 Nonfatal myocardial infarction - % 3.3 2.6 0.52 Q-wave - % 1.1 0.70.39 non-Q-wave - % 2.2 1.9 0.60 Target vessel revascularization - % 3.02.3 0.34

One-year follow-up indicated also that there is no correlation betweenthe presence of the IL-1RN*2 allele and mortality or incidence ofmyocardial infarction after the intervention. During the 1-year period,mortality rate was 2.8% in the combined group of IL-1RN 1/2 and IL-1RN2/2 patients and 2.2% in IL-1 1/1 patients (P=0.42), yielding an oddsratio of 1.28 (95% confidence interval, 0.71-2.29). The incidence ofnonfatal myocardial infarction was 3.5% in IL-1RN*2 allele carriers and3.9% in homozygous carriers of the IL-1RN*1 allele (P=0.54), and therespective odds ratio was 0.86 (0.53-1.4).

IL-1RN Polymorphism and Restenosis After Stenting

Control angiography was performed in 84% of the patients after a medianof 188 days (interquartile range, 171-205 days). The proportion ofpatients with control angiography was similar in the 2 groups defined bythe presence or absence of the IL-1RN*2 allele. Table 7 lists theresults of the quantitative assessment of 6-month angiograms.

TABLE 7 Results at follow-up angiography. IL-1RN 1/2 or 2/2 IL-1RN 1/1(n = 758) (n = 798) P Late lumen loss - mm 1.16 ± 0.82 1.24 ± 0.86 0.07Loss index 0.53 ± 0.38 0.59 ± 0.45 0.009 Diameter stenosis - % 41.8 ±26.2 45.2 ± 28.7 0.015 Restenosis rate - % 30.2 35.6 0.024 Data areproportions or mean ± SDOf note, loss index which reflects the hyperplastic response afterstenting was significantly lower in patients who carried the IL-1RN*2allele. The incidence of angiographic restenosis was also significantlylower in carriers of the IL-1RN*2 allele, with 30.2% vs. 35.6% inpatients of the IL-1RN 1/1 genotype. Thus, the presence of the IL-1RN*2allele was associated with a 22% decrease in restenosis rate (oddsratio, 0.78 [0.63-0.97]; FIG. 9, left panel). Clinical restenosisexpressed as the need for target vessel revascularization was alsosignificantly lower, with 17.7% in IL-1RN*2 allele carriers vs. 22.7% inhomozygous patients for the IL-1RN*1 allele (P=0.026), yielding an oddsratio of 0.73 (0.58-0.92) as shown in FIG. 9, left panel.

Age, gender, the presence or absence of diabetes, smoking habit, reducedleft ventricular function and restenotic lesions, vessel size (allvariables differing in univariate analysis by a P-value 0.30) wereentered into the multivariate model for angiographic restenosis alongwith the presence or absence of the IL-1RN*2 allele. Older age(P=0.005), the presence of diabetes (P<0.001), restenotic lesion(P<0.001) and small vessel size (P<0.001) were independently correlatedwith an increased risk of restenosis. On the opposite, the presence ofthe IL-1RN*2 allele was independently (P<0.001) correlated with adecreased risk for restenosis with an adjusted odds ratio of 0.81(0.71-0.92). In addition, there was a significant interaction betweenthe presence of the IL-1RN*2 allele and age (P=0.009) as reflected by aprogressively stronger protective effect of this allele in youngerpatients.

The results of the analysis in the prespecified subgroup of patients <60years (n=696) are presented in Table 8, FIG. 9, right panel and FIG. 10.During the 1-year follow-up period, 17.1% of the IL-1RN*2 allelecarriers and 24.9% of the homozygous IL-1RN*1 allele carriers neededtarget vessel revascularization (P=0.013). Thus, the presence of theIL-1RN*2 allele was associated with a 37% reduction (odds ratio: 0.63[0.43-0.91]; FIG. 1, right panel) of the need of ischemia-drivenreinterventions. Quantitative angiographic data obtained for the controlstudy at 6 months (performed in 590 or 85% of patients <60 years) aredisplayed in Table 8.

TABLE 8 Results at follow-up angiography in patients <60 years. IL-1RN1/2 or 2/2 IL-1RN 1/1 (n = 273) (n = 317) P Late lumen loss - mm 1.08 ±0.77 1.27 ± 0.93 0.008 Loss index 0.49 ± 0.35 0.59 ± 0.48 0.003 Diameterstenosis - % 39.3 ± 24.1 46.7 ± 30.5 0.001 Restenosis rate - % 25.6 38.5<0.001 Data are proportions or mean ± SDThe incidence of angiographic restenosis was 25.6% in the combined groupof IL-1RN 1/2 and IL-1RN 2/2 patients and 38.5% among IL-1RN 1/1patients (P<0.001), which corresponds to a 45% reduction (odds ratio:0.55 [0.39-0.78]; FIG. 1, right panel). FIG. 2 illustrates the gene doseeffect verified in the subgroup of younger patients. The incidence ofrestenosis decreased progressively with heterozygosity and homozygosityfor the IL-1RN*2 allele. The rate of angiographic restenosis was 38.5%in IL-1RN 1/1 patients, 26.3% in IL-1RN 1/2 patients and 22.4% in IL-1RN2/2 patients (P=0.001, test for trend). The target vesselrevascularization rate was 24.9% in IL-1RN 1/1 patients, 17.9% in IL-1RN1/2 patients and 13.2% in IL-1RN 2/2 patients (P=0.01, test for trend;FIG. 2).

5.3. Example 3 The IL-1 Haplotype Patterns Associated with OcclusiveCardiovascular Disorders and Periodontitis

The association between periodontitis, cardiovascular disease and fourbasic biallelic markers (IL-1A (+4845), IL-1B (+3954), IL-1B (−511), andIL-1RN (+2018)) in the interleukin-1 (IL-1) gene cluster on chromosome 2was investigated.

Two haplotype patterns may be defined by four polymorphic loci in theIL-1 gene cluster as shown in Table 9 (IL-1A(+4845), IL-1B(+3954), IL-1B(−511), IL-1RN (+2018)). One pattern includes allele 2 at both the IL-1A(+4845) and at the IL-1B (+3954) loci. The other pattern includes allele2 at both the IL-1B(−511), and at the IL-1RN (+2018) loci.

TABLE 9 IL-1RN Haplotypes IL-1A (+4845) IL-1B (+3954) IL-1B (−511)(+2018) Pattern 1 Allele 2 Allele 2 Allele 1 Allele 1 Pattern 2 Allele 1Allele 1 Allele 2 Allele 2

The haplotype pattern indicates that when allele 2 is found at onelocus, it is highly likely that it will be found at other loci. Previousdata (Cox et al. (1998) Am. J. Hum. Genet. 62:1180-1188) indicate thatwhen allele 2 is found at the IL-1A (+4845) locus allele 2 will also bepresent at the IL-1B (+3954) locus approximately 80% of the time.Haplotype patterns are relevant only for a single copy of a chromosome.Since there are two copies of chromosome 2 and standard genotypingprocedures are unable to identify on which chromosome copy a specificallele is found, special statistical programs are used to inferhaplotype patterns from the genotype pattern that is determined.

The distribution of these genetic patterns was evaluated in a newpopulation that was part of a study of atherosclerosis (Pankow et al.(1999) The ARIC study. European Atherosclerosis Society Annual Meeting,Abstract, #646). In this population (N=1,368), IL-1A(+4845) genotype 2.2was found in 10.2% of the subjects. However, in the subjects withgenotype IL-1B (+3954)=2.2 (N=95), the IL-1A (+4845) genotype 2.2 wasfound in 71.6% of the subjects. This indicates that allele 2 at IL-1A(+4845) is inherited together with allele 2 at IL-1B (3954) at a muchhigher rate than one would expect given the distribution of each ofthese markers in the population. Similar data exists for allele 2 at the2 loci that are characteristic of Pattern 2. In addition, when genotypePattern 1 is found it is highly unlikely that allele 2 will be presentat either of the loci that are characteristic of the other pattern.

The two genotype patterns are also associated with specific differencesin the functional biology of interleukin-1. For example, peripheralmonocytes from individuals with one or two copies of allele 2 at IL-1B(+3954) produced 2 to 4 times as much IL-1β when stimulated with LPS asmonocytes from individuals who have the genotype pattern IL-1B(+3954)=1.1 (DiGiovini, F S et al. (1995) Cytokine, 7:606). Similar datahave recently been reported for peripheral blood polymorphonuclearleukocytes isolated from individuals with severe periodontitis (Gore, EA et al. (1998) J. Clin. Periodontol., 25:781). In addition gingivalcrevice fluid (GCF) from subjects with the composite genotypesindicative of Pattern 1 have 2 to 3 times higher levels of IL-1β thanGCF from individuals who are negative for those genotypes (Engelbretson,S P et al. (1999) J. Periodontol., in press). There are also dataindicating that for Pattern 2, allele 2 at IL-1RN+2018 is associatedwith decreased levels of IL-1 receptor antagonist protein. Thus, Pattern1 genotypes appear to be associated with increased IL-1 agonists, andPattern 2 appears to be associated with decreased levels of IL-1receptor antagonist.

The composite IL-1 genotypes that are consistent with Pattern 1 areassociated with increased susceptibility to severe adult periodontitis(Kornman, K S et al. (1997), supra; Gore, E A et al. (1998), supra;McGuire, M K et al. (1999) J. Periodontol., in press; McDevitt, M J etal. (1999) J. Periodontol., in press). One aspect of the IL-1 genotypeinfluence on periodontitis appears to be an enhancement of thesubgingival levels of specific bacterial complexes that include acceptedperiodontal pathogens (Socransky, S S et al. (1999) IADR Annual Meeting,Abstract#3600). Pattern 1 genotypes were not, however, associated withincreased risk for occlusive cardiovascular disease. In data from theAtherosclerosis Risk in Communities (ARIC) study that was presented byPankow and co-workers (see Pankow et al., supra), individuals withultrasound measurements of carotid wall intima-medial thickness (IMT)that were indicative of occlusive cardiovascular disorders were comparedto a stratified random control population for IL-1 gene polymorphisms.Neither IL-1A (+4845) or IL-1B (+3954) showed any association with riskfor high IMT.

Genotypes that are characteristic of pattern 2 have recently beenassociated with increased susceptibility to occlusive coronary arterydisease, but not increased risk for periodontitis. In a report oncoronary artery disease, patients with angiographic evidence of coronarystenoses were significantly more likely to be carriers of allele 2 ateither the IL-1RN (+2018) locus or the IL-1B (−511) locus (see Franciset al., supra). Both loci are characteristic of the haplotype Pattern 2.In the ARIC study, as discussed above, carriage of IL-1RN (+2018) allele2 in African-Americans with high IMT measurements was significantlyhigher than ethnically matched controls. In Caucasians with high IMTmeasurements the carriage of one copy of allele 2 at IL-1RN (+2018) wassignificantly greater than in controls, however individuals homozygousat this locus were not different from controls. It should be noted thatthe prevalence of individuals homozygous for allele 2 at IL-1RN (+2018)in Caucasians in the study was substantially lower than that observed inother populations.

When individuals with periodontitis and gingival health were evaluatedfor genotype patterns consistent with Pattern 1 and Pattern 2,individuals with severe adult periodontitis were found to have apredominance of genotypes consistent with Pattern 1, whereas individualswith a healthy periodontal condition had genotype patterns that weredominated by neither Pattern 1 nor Pattern 2. It appears therefore thatIL-1 genotypes consistent with the haplotype Pattern 1 are associatedwith severe periodontitis and plaque fragility disorders and notocclusive cardiovascluar diseases whereas IL-1 genotypes consistent withthe haplotype Pattern 2 are associated with occlusive cardiovasculardiseases but not periodontitis or plaque fragility. One mechanism may bethat IL-1 genotype Pattern 1 directly influences plaque fragility;another mechanism may be that Pattern 1 influences periodontitisdirectly, which may lead to indirect influences on cardiovasculardisease through the periodontal micororganisms found as part of the oralchronic inflammatory process. Another mechanism may be that IL-1genotype Pattern 2 directly influences cardiovascular occlusivedisorders but has no influence on periodontitis. It is thus likely thatIL-1 genetic polymorphisms can influence both cardiovascular disease andsevere periodontitis, by a common underlying mechanism that directlyalters the immunoinflammatory responses in both diseases in an identicalfashion and by an indirect mechanism that enhances the oral bacterialload and then influences cardiovascular disease. The IL-1 genotypes thatare consistent with haplotype Pattern 1 may influence the associationbetween periodontidis and cardiovascular disease in one segment of thepopulation by amplifying both the immuno-inflammatory response and thesubgingival bacterial load.

5.4 Example 4 Genotyping Methods Preparation of DNA

Blood is taken by venipuncture and stored uncoagulated at −20° C. priorto DNA extraction. Ten milliliters of blood are added to 40 ml ofhypotonic red blood cell (RBC) lysis solution (10 mM Tris, 0.32 Sucrose,4 mM MgCl₂, 1% Triton X-100) and mixed by inversion for 4 minutes atroom temperature (RT). Samples are then centrifuged at 1300 g for 15minutes, the supernatant aspirated and discarded, and another 30 ml ofRBC lysis solution added to the cell pellet. Following centrifugation,the pellet is resuspended in 2 ml white blood cell (WBC) lysis solution(0.4 M Tris, 60 mM EDTA, 0.15 M NaCl, 10% SDS) and transferred into afresh 15 ml polypropylene tube. Sodium perchlorate is added at a finalconcentration of 1M and the tubes are first inverted on a rotary mixerfor 15 minutes at RT, then incubated at 65° C. for 25 minutes, beinginverted periodically. After addition of 2 ml of chloroform (stored at−20° C.), samples are mixed for 10 minutes at room temperature and thencentrifuged at 800 G for 3 minutes. At this stage, a very cleardistinction of phases can be obtained using 300 l Nucleon Silicasuspension (Scotlab, UK) and centrifugation at 1400 G for 5 minutes. Theresulting aqueous upper layer is transferred to a fresh 15 mlpolypropylene tube and cold ethanol (stored at −20° C.) is added toprecipitate the DNA. This is spooled out on a glass hook and transferredto a 1.5 ml eppendorf tube containing 500 l TE or sterile water.Following overnight resuspension in TE, genomic DNA yield is calculatedby spectrophotometry at 260 nm. Aliquots of samples are diluted at 100ug/ml, transferred to microtiter containers and stored at 4° C. Stocksare stored at −20° C. for future reference.

5.4.1 Polymerase Chain Reaction

Oligonucleotide primers designed to amplify the relevant region of thegene spanning the polymorphic site (as detailed below) are synthesized,resuspended in Tris-EDTA buffer (TE), and stored at −20° C. as stocksolutions of 200 uM. Aliquots of working solutions (1:1 mixture offorward and reverse, 20 μM of each in water) are prepared in advance.

Typically, PCR reaction mixtures are prepared as detailed below.

Final Stock Concentration Volume Concentration Sterile H₂0 29.5 μl10xPCR buffer 200 mM Tris-HCl 5.00 μl 20 mM Tris-HCl, (pH 8.4) MgCl₂ 50mM 1.75 μl 1.75 mM dNTP mix 10 mM of each 4.00 μl 0.2 mM of each primerforward 20 uM 2.5 μl 1 uM prime reverse 20 uM 2.5 μl 1 uM Tag polymerase5 U/μl 0.25 μl 1.25 units/50 μl Detergent (eg W-1, 1% 2.5 μl 0.05%Gibco) Template 200 ng/μl 2.00 μl 2 ng/l Final Volume 50.00 μl

DNA template is dotted at the bottom of 0.2 ml tubes or microwells. Thesame volume of water or negative control DNA is also randomly tested. Amaster-mix (including all reagents except templates) is prepared andadded to the wells or tubes, and samples are transferred to thethermocycler for PCR.

PCR can be performed in 0.5 ml tubes, 0.2 ml tubes or microwells,according to the thermocycler available. The reaction mixture isoverlaid with mineral oil if a heated lid (to prevent evaporation) isnot available.

5.4.2 Restriction Enzyme Digestion

A master mix of restriction enzyme buffer and enzyme is prepared andaliquotted in suitable volumes in fresh microwells. Digestion is carriedout with an oil overlay or capped microtubes at the appropriatetemperature for the enzyme on a dry block.

Restriction buffer dilutions are calculated on the whole reaction volume(i.e. ignoring salt concentrations of PCR buffer). Restriction enzymesare used 3-5 times in excess of the recommended concentration tocompensate for the unfavorable buffer conditions and to ensure completedigestion.

5.4.3 Electrophoresis

Polyacrylamide-gel electrophoresis (PAGE) of the PCR sample is carriedout in Tris-HCl-EDTA buffer and at constant voltage. Depending on thesize discrimination need, different PAGE conditions are used (9 to 12%acrylamide, 1.5 mm×200) and different DNA size marker (X174-Hae III or X174-Hinf 1). A 2% agarose horizontal gel can be used for genotyping theIL-1RN (VNTR) marker.

5.4.4 Allele Detection Methods

The following Table 10 provides methods for detecting particular allelesthat are associated with the existence of or susceptibility todeveloping restenosis.

TABLE 10 IL-1A (+4845) 5′ Primer ATG.GTT.TTA.GAA.ATC.ATC.AAG.CCT.AGG.GCA(+4814/+4843) (SEQ ID No. 1) 3′ PrimerAAT.GAA.AGG.AGG.GGA.GGA.TGA.CAG.AAA.TGT (+5015/+5044) (SEQ ID No. 2) PCRMgCl₂ is used at 1 mM final, and PCR primers at 0.8 mM. DMSO isConditions added at 5%, DNA template at 150 ng/50 ml, and TaqMan 1.25u/50 μl. Cycling 1X [95° C. 1 min.]; 35X [94° C. 1 min., 56° C. 1 min.,72° C. 2 min.]; 1X conditions [72° C. 5 min.]; 4° C. Analysis Cleavagewith 2.5 units of Fnu4H1 in addition to 2 ml of the specific 10restriction buffer at 37° C. overnight, followed by 9% PAGE analysisyields a constant band of 76 bp (absence indicates incomplete digestion)and two further bands of 29 and 124 bp (allele 1) , or a single band of153 bp (allele 2). Allele frequencies in North British Caucasianpopulation are 0.71 and 0.29. Reference Gubler, et al.(1989)Interleukin, inflammation and disease (Bomford and Henderson, eds.)p.31-45, Elsevier publishers; and Van den velden and Reitsma (1993) HumMol Genetics 2: 1753-50). GenBank Accession No. X03833. IL1B (-511)5′ Primer TGG.CAT.TGA.TCT.GGT.TCA.TC (−702/−682) (SEQ ID No: 3)3′ Primer GTT.TAG.GAA.TCT.TCC.CAC.TT (−417/−397) (SEQ ID No: 4) PCR 50mM KCl, 10 mM Tris-HCl, pH 9.0, 1.5 mM MgCl₂, 200 mM dNTPs, Conditions25 ng primers, 50 ng template, 0.004% W-1 (Gibco-BRL), 0.2 U Taqpolymerase, 50 μl total volume Cycling 1X [95° C. 2 min.]; 35X [95° C. 1min., 53° C. 1 min., 72° C. 1 min.]; 1x conditions [72° C., 5 min.];4° C. Analysis Each PCR reaction is divided into two 25 μl aliquots: oneis added of 3 units of Ava I restriction endonuclease, the other 3.7units of Bsu 36 I, in addition to 3 μl of the specific 10x restrictionbuffer. Incubation is at 37° C. overnight. Electrophoresis is by PAGE9%. Cleavage with Ava I and Bsu 36I. Allele 1 (C) produces 190 and 114bp fragments when digested with Ava I and a 304 bp fragment whendigested with Bsu 36I. Allele 2 (T) produces a 304 bp fragment whendigested with Ava I and 190 and 114 bp fragments when digested with Bsu36I. The restriction pattern obtained should be the inverse in the twoaliquots (identifying homozygotyes) or identical (heterozygotes).Frequencies in North British Caucasian population are 0.61 and 0.39 forallele 1 and 2 respectively. Reference diGiovine, Hum. Molec. Genet.,1(6): 450 (1992); Clark, et al., Nucl. Acids. Res., 14: 7897-7914 (1986)[published erratum appears in Nucleic Acids Res., 15(2): 868 (1987)];GenBank Accession No. X04500. IL-1B (+3954) 5′ PrimerCTC.AGG.TGT.CCT.CGA.AGA.AAT.CAA.A (+3844/+3868) (SEQ ID No: 5) 3′ PrimerGCT.TTT.TTG.CTG.TGA.GTC.CCG (+4017/+4037) (SEQ ID No: 6) PCR 50 mM KCl,10 mM Tris-HCl, pH 9.0, 1.5 mM MgCl₂,200 mM dNTPs, Conditions 25 ngprimers, 50 ng template, 0.004% W-1 (Gibco-BRL), 0.2 U Taq polymerase,50 μl total volume Cycling 1X [95° C. 2 min.]; 35 X [95° C. 1 min.;67.5° C. 1 min., 72° C. 1 min]; conditions 1X [72° C., 5 min]; 4° C.Analysis Each PCR reaction is added of 10 u of Taq 1 restrictionendonuclease in addition to 3 μl of the specific 10x restriction buffer.Incubation is at 65° C. overnight. Electrophoresis is by PAGE 9%.Following digestion with Taq I, Allele 1 produces 97, 85 and 12 bpfragments; Allele 2 produces 182 and 12 bp fragments. The absence of the12 bp band indicates incomplete digestion. Frequencies in a NorthBritish Caucasian population are 0.82 (allele 1) and 0.18 (allele 2).For 90% power at 0.05 level of significance in a similar genetic pool,408 cases should be studied to detect 1.5 fold increase in thefrequency, or 333 for 0.1 absolute increase in frequency. Reference diGiovine, et al. Cytokine 7(6): 606 (1995) IL-1RN (VNTR) 5′ PrimerCTC.AGC.AAC.ACT.CCT.AT (+2879/+2895) (SEQ ID NO. 7) 3′ PrimerTCC.TGG.TCT.GCA.GGT.AA (+3274/+3290) (SEQ ID NO. 8) PCR 50 mM KCl, 10 mMTris-HCl pH 9.0, 1.7 mM MgCl₂, 200 mM dNTPs, Conditions 25 ng primers,50 ng template, 0.004% W-1 (Gibco-BRL) 0.2 u Taq polymerase Cycling 1 X[96° C. for 1 min.]; 30 X [94° C. for 1 min., 60° C. for 1 min., 70°conditions C. for 1 min.]; 1 [70° C. for 2 min.]. Analysis The variablenumber of tandem repeats (VNTR) in intron 2 of IL1-RN corresponds to avariable number (2 to 6) of an 86 bp repeat and so the PCR product sizesare a direct indication of the number of repeats. Electrophoresis is by2% agarose, 90V, 30 min. Allele 1 4 repeats 412 bp PCR product Allele 22 repeats 240 bp PCR product Allele 3 3 repeats 326 bp PCR productAllele 4 5 repeats 498 bp PCR product Allele 5 6 repeats 584 bp PCRproduct Frequencis in a North British Caucasian population for the fourmost frequent alleles are 0.734, 0.241, 0.021 and 0.004. ReferenceSteinkasserer et al. (1991) Nucleic Acids Research 19: 5090-95; Tarlow,et al., Hum. Genet. 91: 403-4 (1993) IL-1RN (+2018) 5′ PrimerCTA.TCT.GAG.GAA.CAA.CCA.ACT.AGT.AGC-3′ (+1992/+2017) (SEQ ID No. 9)3′ Primer TAG.GAC.ATT.GCA.CCT.AGG.GTT.TGT-3′ (+2135/2158) (SEQ ID No.10) PCR Each PCR reaction is divided in two 25 μl aliquots; to one isadded 5 Conditions Units of Alu I, the other 5 Units of Msp I, inaddition to 3 μl of the specific 10X restriction buffer. Incubation is a37° C. overnight. Electrophoresis is by PAGE 9%. Cycling 1 X [96° C. for1 min]; 35 X [94° C. for 1, min., 57° C. for 1 min 70° C. conditions for2 min.]; 1 X [70° for 5 min.]; 4° C. Allele The above described PCRprimers incorporate mismatches to the Detection genomic sequence so asto engineer two different restriction sites on the alleles. The twoalleles are 100% in linkage disequilibrium with the two most frequentalleles of IL-1RN (VNTR). Alu I will produce 126 + 28 bp fragments forAllele 1, while it does not digest Allele 2 (154 bp). Msp I will produce125 + 29 bp with Allele 2, while Allele 1 is uncut (154 bp). Hence thetwo reactions (separated side by side in PAGE) will give invertedpatterns of digestion for homozygote individuals, and identical patternsin heterozygotes. Allelic frequencies in a North British Caucasionpopulation are 0.74 and 0.26. For 90% power at 0.05 level ofsignificance in a similar genetic pool, 251 cases should be studied todetect 1.5 fold increase in frequency, or 420 for 0.1 absolute increasein frequency. Reference Clay, et al.(1996) Hum. Genet. 97: 723-26.

Results: Typing of additional numbers of individuals is required tobring the results to significance, but preliminary results indicate thatallele 2 of the 4845, −511, +3954 and VNTR markers in the IL-1RN genewill be over-represented in restenosis. It is predicted that individualswith at least one copy of allele 2 from one of the above markers aremore likely to have restenosis than those who are negative for allele 2.Individuals who are homozygous for any of these alleles, or have allele2 from more than one marker are estimated to have even higher risk forrestenosis.

5.5 Example 5

In this example, the preparation of template DNA is described. PCR-basedgenotyping does not require particularly high-MW DNA (<20 Kb DNA isoften an excellent template). As 100 ng genomic DNA is more thansufficient for single-copy gene amplification, direct amplification fromdried blood spots or cell lysates can be used for genotyping, and two ofthe protocols that we have used are here described below.

However, if DNA banks need to be established for population studieswhere DNA needs to be stored for future reference or genotyping atdifferent loci, or where genomic Southern blotting might be needed, goodquality high-MW genomic DNA needs to be extracted. Basic buffers and thecomposition of chemical solutions can be found in major protocoltextbooks (Sambrook et al. (1989) Molecular cloning: a laboratorymanual, Cold Spring Harbor Press; Ausubel and Frederick (1994) Currentprotocols in molecular biology, John Wiley and Sons).

Sample DNA can also be obtained from dried blood spots. Such a means ofsample collection (Guthrie spots) has been used for many years inneonatal diagnosis of phenylketonuria. In the last few years dried bloodspots have proved useful in PCR-based diagnostics (Raskin et al. (1991)Am J Hum Genet 49: 320-29). Uncoagulated blood is spotted evenly using asterile Pasteur pipette onto a clean sheet of filter paper. This is leftto dry overnight in a clean area (physically isolated from post-PCRevents) and stored subsequently at room temperature.

For PCR, a mastermix is prepared as described later in this chapter,where Taq polymerase is omitted. This is aliquotted in reaction tubes,and approximately 1 mm² of the blood spot is cut out and placed into thereaction mix. This is overlaid with 40 μl mineral oil. The lid of eachtube is pierced with a sterile needle, and samples are then heated at98° C. for 15 minutes. Following cooling for a few minutes, Taqpolymerase is added and standard PCR cycling follows.

Sample DNA can also be obtained from cell lysates. White blood cells,buccal cells or homogenised tissue is suspended in PK buffer (0.1M NaCl,10 mM Tris-HCl, 25 mM EDTA, 0.5% SDS pH 8.0, 0.1 mg/ml fresh ProteinaseK) and incubated on a tumbler at 37° C. for 1 hour. Samples are heatedat 95° C. for 10 mins, spun at 13,000 rpm in a microfuge andsupernatants stored at −20° C. prior to PCR. For higher quality DNA, aphenol/chloroform extraction followed by ethanol precipitation can beadded.

Sample genomic DNA can also be obtained from whole blood. Blood is takenby venepuncture and stored uncoagulated at −20° C. prior to DNAextraction. When possible, we prefer to collect two 10 ml samples,extract DNA form the first and keep the second for future reference. Tenmilliliters of blood are added to 40 ml of hypotonic red blood cell(RBC) lysis solution (10 mM Tris-HCl, 0.32 Sucrose, 4 mM MgCl₂, 1%Triton X-100) and mixed by inversion for 4 minutes at room temperature.Samples are then centrifuged at 1300 g for 15 minutes, the supernatantaspirated and discarded, and another 30 ml of RBC lysis solution addedto the cell pellet. Following centrifugation, the pellet is resuspendedin 2 ml white blood cell (WBC) lysis solution (0.4M Tris-HCl, 60 mMEDTA, 0.15M NaCl, 10% SDS) and transferred into a fresh 15 mlpolypropylene tube. Sodium perchlorate is added at a final concentrationof 1M and the tubes are first inverted on a rotary mixer for 15 minutesat room temperature (RT), then incubated at 65° C. for 25 minutes, beinginverted periodically. After addition of 2 ml of chloroform (stored at−20° C.), samples are mixed for 10 minutes at room temperature and thencentrifuged at 800 g for 3 minutes. At this stage a very cleardistinction of phases can be obtained using 300 μl Nucleon Silicasuspension (Scotlab, UK) and centrifugation at 1400 G for 5 minutes. Theresulting aqueous upper layer is transferred to a fresh 15 mlpolypropylene tube and cold ethanol (stored at −20° C.) is added toprecipitate the DNA. This is spooled out on a glass hook and transferredto a 1.5 ml eppendorf tube or containing 500 μl TE or sterile water.Following overnight resuspension in TE, genomic DNA yield is calculatedby spectrophotometry at 260 nm. Aliquots of samples are diluted at 100μg/ml, transferred to microtiter containers and stored at 4° C. Stocksare stored at −20° C. for future reference.

5.6 Example 6

In this example, the conditions for conducting appropriate polymerasechain reactions on the collected samples are described. Oligonucleotideprimers designed to amplify the relevant region of the gene spanning thepolymorphic site (as detailed below) are synthesised, resuspended inTris-HCl-EDTA buffer (TE) and stored at −20° C. as stock solutions of200 μM. Aliquots of working solutions (1:1 mixture of forward andreverse, 20 μM of each in water) are prepared in advance of theexperiment. Typically PCR reaction mixtures are prepared as detailedbelow. Divergence from the scheme below can be made for each specificprotocol.

Stock Final Concentration Volume Concentration Sterile H₂0 29.5 μl10xPCR buffer 200 mM Tris-HCl (pH 5.00 μl 20 mM Tris-HCl, 8.4), 500 mMKCl 50 mM KCl MgCl₂ 50 mM 1.75 μl 1.75 mM dNTP mix 10 mM of each 4.00 μl0.2 mM of each primer forward 20 μM 2.5 μl 1 μM primer reverse 20 μM 2.5μl 1 μM Taq polymerase 5 U/μl 0.25 μl 1.25 units/50 μl Detergent (eg 1%2.5 μl 0.05% W-1 Gibco) Template 200 ng/μl 2.00 μl 2 ng/μl Final volume50.00 μl

DNA template is dotted at the bottom of 0.2 ml tubes or microwells. Thesame volume of water or negative control DNA is also randomly tested. Amaster-mix (including all reagents except templates) is prepared andadded to the wells or tubes, and samples are transferred to thethermocycler for PCR.

PCR can be performed in 0.5 ml tubes, 0.2 ml tubes or microwells,according to the thermocycler available and to the needs of the project.The reaction mixture is overlaid with mineral oil if a heated lid (toprevent evaporation) is not available. We use 96-well formatmicroplates, because they allow use of multichannel pipettes both fortransfer of template DNA (stored in 1 ml/microwell plates) and fordispensing of the reaction mastermix.

5.7 Example 7

In this example, the conditions for conducting appropriate polymerasechain reactions on the collected samples are described. A master mix ofrestriction enzyme buffer and enzyme is prepared and aliquotted insuitable volumes in fresh microwells. We use a multichannel pipette totransfer and mix 25-30 μl of PCR product in the microwells. Digestion iscarried out with an oil overlay or capped microtubes at the appropriatetemperature for the enzyme on a dry block. Restriction buffer dilutionsare calculated on the whole reaction volume (i.e. ignoring saltconcentrations of PCR buffer). Restriction enzymes are used 3-5 times inexcess of the recommended concentration, to compensate for theunfavorable buffer conditions and to ensure complete digestion.

5.8 Example 8

In this example, the conditions for conducting gel electorphoresisanalysis of the products of pcr amplification and restrictionendonuclease digestion are considered. Polyacrylamide-gelelectrophoresis (PAGE) of 20-40 μl PCR sample is carried out inTris-HCl-EDTA buffer and at constant voltage. Depending on the sizediscrimination needed, different PAGE conditions are used (9 to 12%acrylamide, 1.5 mm×200) and different DNA size markers ((φX174-Hae IIIor φX 174-HinfI). A 2% agarose horizontal gel can be used for IL—1RN(VNTR).

5.9 Example 9

In this example, quality controls for these genotyping protocols areconsidered. Incomplete digestion is the most common cause of mis-typingin PCR-RFLP genotyping methods. Most of the protocols described hereinare based on a double-cut strategy, for which either a secondrestriction cutting site is used for digestion control on the diagnosticcleavage, or one enzyme cuts one allelic DNA form, and a differentenzyme cuts the other allele. In this case each reaction is the controlfor the other. PCR conditions are tested (and, if necessary,re-optimised) for each DNA preparation not performed in our laboratory.Template DNA quality is assessed by spectrophotometry and by gelelectrophoresis.

The possibility of cross-contamination is very high in PCR-basedtechniques. Although the genotyping is physically separated from any labwhere relevant cloned fragments are being handled, it is still possibleto have PCR-product carryover from previous experiments (from labcoat,hair, skin, etc.). A “PCR-carryover prevention kit” is available fromPerkin-Elmer. This is based on UNG treatment of samples prior to PCR,which will cleave all dUTP-containing DNA. As all PCRs are performedusing dUTP instead of dTTP, all previous PCR products, but not nativetemplates, will be cleaved in this digestion step. This enzyme isinactivated by the first temperature ramping (94° C.) and thereforenormal PCR can take place without UNG activity. If laboratories do notuse this system (which is expensive), there are stringent rules that canbe used to reduce the risk of artefacts due to contamination.

5.10 Example 10

In this example, the prevention of contamination in these genotypingprotocols is considered. Incomplete digestion is the most common causeof mis-typing in PCR-RFLP genotyping methods.

Laboratories are divided into GREEN (Pre-PCR) and RED (Post-PCR) areas.All laboratories have dedicated white coats, and workers are encouragedto change lab gloves as frequently as possible. GREEN laboratories havethe most stringent requirements. Only goods coming from other greenareas can enter, anything (equipment included) that leaves them cannotre-enter. These usually include a store-room, a “sample reception” area,a “clean DNA room” (where DNA extraction and PCR preparation areperformed) and offices. RED laboratories have open access, but materialand equipment can only move to other red areas or disposed of in bagsfor autoclaving or incineration. Red areas are where PCR andelectrophoresis take place. Results and images are stored in computerfiles and transferred to the offices by local network.

All PCR's carry 10% negative controls which are randomly placed withinthe experiment. These are routinely represented by water controls. Inthe case of amplicards, negative controls are represented also byfragments (2-3-mm²) of paper from the edge of the card. For human bloodDNA preparations, murine T cell lysates are extracted at the same timeas each new batch of frozen blood, and resulting DNA used as negativecontrol.

5.11 Example 11

In this example, the design of human polymorphic marker associationstudies are examined and the resulting data is analyzed. Traditionalparametric analyses (requiring the specification of a distributionand/or the mode of inheritance) have been used successfully to locategenes for monogenic diseases following simple Mendelian modes ofinheritances. More commonly used in the genetic analysis of complexdiseases are non-parametric methods since these work independently ofinheritance specifications, and are generally more powerful thanparametric methods when parameters are mis-specified. The choice ofmethod of analysis depends on whether the investigator wishes to performa whole genome screen or use a candidate gene approach, since certainmethods are best suited to just one of these two approaches or tospecific pedigree structures. The following sections contain an outlineof most commonly used non-parametric methods of analysis and theirsuitability to the candidate gene approach.

An allele at a certain locus is said to be associated with a disease ifthe frequency for that allele is significantly increased in the diseasepopulation over that of the normal healthy control population. Trueassociations are due to linkage disequilibrium, where the diseasecausing allele at the ‘disease’ locus remains on the same haplotype asthose alleles which were present at closely flanking loci when theancestral mutation occurred. Thus, the frequency of any allele on the‘disease haplotype’ (including, of course, the disease allele itself)will be increased in the disease population. Recombination overextremely small distances is very low, but as the time from theancestral mutation increases, the distance over which linkagedisequilibrium acts decreases reducing the length of the ‘diseasehaplotype’. It is therefore easier to detect association in young,isolated populations with a single founder mutation effect where linkageextends over larger distances, than in large mixed populations.

Association studies are at present only suited to the candidate geneapproach due to the small distances over which associations aredetectable. In the future it is proposed that genome-wide associationstudies will be performed using several biallelic markers in every gene.Care must be taken when selecting the disease population in anassociation study, since spurious positive results may occur as anartefact of population admixture. It is usually advisable to investigatewithin a single ethnic group, since allele frequencies may vary betweendifferent groups. Similarly, if a control population is needed, it mustbe matched to the disease group for ethnicity, and ideally sex and age.

Case control studies can be performed for both qualitative andquantitative phenotypes. Obvious advantages of this approach include theease of collection of large populations, the possibility of recruitmentof patients with “early disease” phenotypes, and the possibility ofanalyzing late-onset diseases, where parental DNA may not be available.

For qualitative phenotypic studies, the candidate gene locus, allelefrequencies or alternatively genotype frequencies, within the diseaseand control populations are calculated. The analysis is simple,comprising of a 2×n contingency table (n denoting the number ofcategories, 2 for allele frequencies or 3 for genotypes at a bialleliclocus), which a chi-square test may be used to determine whether theproportions differ significantly between the disease and controlpopulations.

For quantitative phenotypic studies looking for a disease susceptibilityallele, the individuals in both populations are first phenotypedquantitatively (usually the disease is classified as attaining a certainthreshold value, therefore the unaffected controls are individualsfailing below this). All individuals are then subdivided into the three(or more) genotypes. If an allele responsible for the inflated phenotypevalue of the diseased individuals exists, it would be expected thatthese individuals carry at least, one copy of it. Thus the median ofthese genotype groups would be higher than those of the non-carriergroups. The non-parametric test involves testing for significantdifference between the medians of the different genotype (or carriage)groups. This may be done via a Mann-Whitney test (for 2 groups), or aKruskall-Wallis (for >2 groups), although several other tests alsoexist. In exactly the same way, this type of analysis may also beperformed solely within the disease group.

For use of qualitative traits in studies employing more than one IL-1polymorphic locus, the simple one locus case-control analysis can beextended to one involving several loci (given a sufficient sample size).In a similar way, a larger contingency table can be calculated, withgroups corresponding now to composite genotypes. As before, achi-squared statistic can be calculated. With these large contingencytables, it is likely that the validity of the chi-square test isviolated (<80% of expected values >5, and expected values <1). Withsmaller contingency tables, the usual remedy to violations of validityis to use Fishers Exact test, but in this larger case, it is not viable.Instead a null distribution for the evaluated chi-square statistic issimulated, and significance assessed from this. This test has been namedthe Monte Carlo Composite Genotype (MCCG) test.

5.12 Example 12

In this example, haplotype relative risk (HRR) analysis is discussed.This analysis is only suitable for qualitative traits (quantitativetraits may be used if, dichotomised), and as with all association tests,the candidate gene approach. Haplotype analysis investigates theassociation between specific genetic markers for diseases and the way aset of markers may influence the outcome of the disease. Analyzing therelationship between specific genetic markers and disease is anextremely complex process. The analysis needs to take into account (i)the relation between genetic markers in neighboring genes, (ii) the waythe polymorphic markers affect expression of the gene in question, (iii)the distribution of the genetic markers for a specific polymorphism overboth chromosomes, and (iv) the way the expressed gene product(s) affectthe disease process.

The relationship between these factors can be identified by statisticalequations that look at multipoint linkage analysis,transmission/disequilibrium test (TDT), multipoint quantitative traitloci (QTL) analysis, identity-by-state (IBS), identity-by-descent (IBD),and grouping of multiallelic markers for biological functions related todisease. This approach has been described by Camp ((1997) AmericanJournal of Human Genetics 61: 1424-30); Cox et al ((1998) AmericanJournal of Human Genetics 62: 1180-88); and Almasy and Blangero ((1998)American Journal of Human Genetics 62: 1198-1211).

To perform a HRR analysis (Falk et al. (1987) Ann Hum Genet. 51: 27-233)nuclear families with affected offspring are needed. This type ofanalysis uses an artificial internal control, and therefore the problemof collecting an independent matched control population is removed. Theparents and affected offspring are genotyped. It is then establishedwhich parental alleles were passed on to the affected offspring andwhich were not. From this the transmitted genotype and thenon-transmitted genotype (internal control) are determined and recordedin the transmitted and non-transmitted groups, respectively. The twogroups are then tested for significant differences in the proportions oftheir genotypes.

5.13 Example 13

In this example, the transmission/disequilibrium test (TDT) isdiscussed. This analysis is suitable for qualitative traits investigatedusing a candidate gene approach. Nuclear families are needed, includingat least one parent, all affected offspring, and if possible anunaffected sibling.

The TDT (Spielman et al. (1993) Am J Hum Genet. 52: 506-16) is a testfor both association and for linkage, more specifically, it tests forlinkage in the presence of association. Thus, if association does notexist at the locus of interest, linkage will not be detected even if itexists. It is for this reason that the test has been included in thissection. It may be used as an initial test, but is more commonly usedwhen tentative evidence for association has already been identified. Inthis case, a positive result will not only confirm the initialassociation, but also provide evidence for linkage.

All parents and affected offspring are genotyped. Only parentsheterozygous for the allele of interest may be used in the analysis. Ifthe allele of interest is, or is linked to, the disease allele, thetransmission rate for that allele from heterozygous parents to theiraffected offspring should be elevated. To test if the transmission rateof the allele of interest is significantly elevated, the number of timesit is transmitted, b, and the number of times other alleles aretransmitted, c, are counted. The squared difference of b and c dividedby their sum provides a statistic that follows a chi-square distributionwith one degree of freedom, and can thus be assessed for significantdeviation from the expected under no association or linkage. It is oftenadvised to repeat this procedure using the unaffected offspring from thesame parents to rule out the possibility of a spurious result due tobiased meioses.

The TDT may also be used once linkage on a coarse scale has been shownto provide the fine scale mapping that is necessary to pin-point moreaccurately the disease locus. Of course, these tests are only valid whenassociations within the area also exist.

5.14 Example 14

In this example, the non-parametric linkage analysis is discussed.Non-parametric linkage analysis methods (such as Affected Sib-Pairanalysis, the Haseman-Elston method and Variance Component Method) arebased on the allele sharing status of affected relative pairs, usuallysibs. These methods are suitable for whole genome screens (commonly doneat 10 cM intervals) and also a candidate gene approach (although forfine localisation alternative methods such as the TDT (section 4.2.1.3)should be used).

5.15 Example 15

In this example the analysis of significance and power of the data isexamined. Throughout this section, evidence strong enough to suggestassociation or linkage has been termed significant. The significancelevel of a test is left to the discretion of the investigator, butconventionally a 5% significance level is used. This means that it isaccepted that there is enough evidence to suggest an association (orlinkage) if the result would have occurred only 1 in 20 (0.05) times bychance in data where no association (linkage) existed, that is, there isonly a 0.05 chance that the result is a false-positive. For each test ap-value may be calculated which indicates the probability of the resultoccurring by chance. In a single test, if this value is less than 0.05then significant evidence may be claimed. This concept becomes morecomplicated when multiple, independent tests are performed. For example,if two tests were performed, and each was tested at the 5% level ofsignificance, overall there is a 2 in 20 (0.1) chance of at least oneresult being a false-positive. Thus, for two independent tests, tomaintain an overall significance level of 0.05 (0.05 chance of at leastone test being a false positive) either the individual significancelevel for each test must be lowered to 0.05/2=0.025, or the p-valuesdoubled before assessing the result. This method of correction is calledthe Bonferroni correction. More generally, if n independent tests werecarried out, each individual test should be tested at the 0.05/n level,or alternatively, every p-value multiplied by n before assessing theresults. With non-independent tests, however, the Bonferroni correctionmay be too conservative.

Many investigators may find that they lose their potential significancesthrough the dilution of p-values due to the correction criteria formultiple tests. Unfortunately these corrections are necessary forstatistical correctness and cannot be discarded. However, if the resultsfrom the first set of observations are real, a second replication sampleneed only test those interesting results found from the first. Thisreduces the number of tests necessary on the second set of observationsand thus reduces the dilution, increasing the chance of maintaining thestatistical significance that may have been lost the first time. Forcomplex diseases where there are so many questions to be answered it isperhaps unreasonable to expect that a single sample would be sufficient,and instead anticipate the necessity for a two-stage analysis andprepare accordingly. This is especially true for whole genome screenswhere the corrections necessary are massive. Lander et al. ((1995)Nature Genet. 11: 241-7) list sensible guidelines for claimingsignificance in linkage analyses, specifically in the case of genomescreens.

Along with significance, a second, and equally important issue is thatof power, the ability to pick up significant evidence where it actuallyexists. Given the phenotype, data structure and number of observations,it is important to choose the method of analysis which is most likely todetermine associations or linkages if they exist. In fact, it isadvisable that in the planning stages of these studies the number ofobservations that are necessary to reach a predetermined power level arecalculated. Unfortunately, this task is not as simple as it sounds,since power depends on several factors, of which some may be unknown,for example, allele frequencies, marker informativeness, familialclustering of the disease, recombination between marker and diseaselocus. Even if these factors are known, the power cannot be explicitlycalculated for some methods, and instead empirical powers must be workedout via simulations.

There is no clear answer to which analyses should be done in differentsituations because of the many variables that are involved. However, itis strongly advisable to make the most informed choice possible, usingprevious work that has been done, to increase the chances of detectionand location of genes responsible, or involved in complex diseases.

1. A method for determining whether a subject has or is predisposed to developing an arterial restenosis, comprising detecting a restenosis associated allele in a nucleic acid sample from the subject, wherein detection of the restenosis allele indicates that the subject has or is predisposed to the development of a restenosis.
 2. A method of claim 1, wherein the restenosis allele is selected from the group consisting of allele 1 of any of the following markers: IL-1A (+4845), IL-1B (−511), IL-1B (+3954) and IL-1RN (+2018) or an allele in linkage disequilibrium therewith.
 3. A method of claim 1, wherein said detecting step is selected from the group consisting of: a) allele specific oligonucleotide hybridization; b) size analysis; c) sequencing; d) hybridization; e) 5′ nuclease digestion; f) single-stranded conformation polymorphism; g) allele specific hybridization; h) primer specific extension; and j) oligonucleotide ligation assay.
 4. A method of claim 1, wherein prior to or in conjunction with detection, the nucleic acid sample is subject to an amplification step.
 5. A method of claim 2, wherein said amplification step employs a primer pair selected from the group consisting of any of SEQ ID Nos. 1 and 2; 3 and 4; 5 and 6; 7 and 8; 9 and 10; 11 and 12; and 13 and 13 and
 14. 6. A method of claim 3, wherein said size analysis is preceded by a restriction enzyme digestion.
 7. A method of claim 6, wherein said restriction enzyme digestion uses a restriction enzyme selected from the group consisting of Alu I, Msp I, Nco I, Fnu 4HI, Ava I, Bsu36 I, and Taq I.
 8. A kit for determining the existence of or a susceptibility to developing a restenosis in a subject, said kit comprising a first primer oligonucleotide that hybridizes 5′ or 3′ to an allele selected from the group consisting of allele 1 of any of the following markers: IL-LA (+4845), IL-1B (−511), IL-1B (+3954), IL-1RN (VNTR) and IL-1RN (+2018) or an allele in linkage disequilibrium therewith.
 9. A kit of claim 8, which additionally comprises a second primer oligonucleotide that hybridizes either 3′ or 5′ respectively to the allele so that the allele can be amplified.
 10. A kit of claim 9, wherein said first primer and said second primer hybridize to a region in the range of between about 50 and about 1000 base pairs.
 11. A kit of claim 8, wherein said primer is selected from the group consisting of any of SEQ ID Nos. 1-14.
 12. A kit of claim 8, which additionally comprises a detection means.
 13. A kit of claim 12, wherein the detection means is selected from the group consisting of: a) allele specific oligonucleotide hybridization; b) size analysis; c) sequencing; d) hybridization; e) 5′ nuclease digestion; f) single-stranded conformation polymorphism; g) allele specific hybridization; h) primer specific extension; and j) oligonucleotide ligation assay.
 14. A kit of claim 8, which additionally comprises an amplification means.
 15. A kit of claim 8, which further comprises a control.
 16. A method for selecting an appropriate therapeutic for an individual that has or is predisposed to developing a restenosis, comprising the steps of: detecting whether the subject contains a restenosis associated allele and selecting a therapeutic that compensates for a restenosis causative functional mutation that is in linkage disequilibrium with the restenosis associated allele.
 17. A method of claim 16, wherein said detecting is performed using a technique selected from the group consisting of: a) allele specific oligonucleotide hybridization; b) size analysis; c) sequencing; d) hybridization; e) 5′ nuclease digestion; f) single-stranded conformation polymorphism; g) allele specific hybridization; h) primer specific extension; and j) oligonucleotide ligation assay.
 18. A method of claim 16, wherein prior to or in conjunction with detecting, the nucleic acid sample is subjected to an amplification step.
 19. A method of claim 18, wherein said amplification step employs a primer selected from the group consisting of SEQ ID Nos. 1-14.
 20. A method of claim 17, wherein said size analysis is preceded by a restriction enzyme digestion.
 21. A method of claim 20, wherein said restriction enzyme digestion uses a restriction enzyme selected from the group consisting of Alu I, Msp I, Nco I, Fnu 4HI, Ava I, Bsu 36 I, and Taq I.
 22. A method of claim 21, wherein the restenosis therapeutic is selected from the group consisting of: an agent that suppresses the development of a hyperplasia and an agent that directly inhibits cellular growth.
 23. A method of claim 22, wherein the agent that suppresses the development of a hyperplasia is selected from the group consisting of a lipid lowering drug, an antiplatelet agent, an anti-inflammatory agent, an antihypertensive agent and an anticoagulant.
 24. A method of claim 21, wherein the restenosis therapeutic is a modulator of an IL-1 activity.
 25. A method of claim 24, wherein the IL-1 activity is IL-1α.
 26. A method of claim 24, wherein the IL-1 activity is IL-1β.
 27. A method of claim 24, wherein the IL-1 activity is IL-1RN.
 28. A method of claim 24, wherein the modulator of an IL-1 activity is a protein, peptide, peptidomimetic, small molecule, nucleic acid or a nutraceutical.
 29. A method of claim 24, wherein the modulator is an agonist.
 30. A method of claim 24, wherein the modulator is an antagonist.
 31. A method of claim 16, wherein the restenosis associated allele is selected from the group consisting of: allele 1 of any of the following markers: IL-1A (+4845), IL-1B (−511), IL-1B (+3954) and IL-1RN (+2018) or an allele in linkage disequilibrium therewith.
 32. A method of claim 16, wherein the restenosis causative functional mutation is an allele of IL-1B (+6912), IL-1B (−511) or IL-1RN (+2018).
 33. A method for determining the effectiveness of treating a subject that has or is predisposed to developing restenosis with a particular dose of a restenosis therapeutic, comprising the steps of: a) detecting the level, amount or activity of an IL-1 protein; or an IL-1 mRNA or DNA in a sample obtained from a subject; (b) administering the particular dose of the particular therapeutic to the subject; detecting the level, amount or activity of an IL-1 protein; or an IL-1 mRNA or DNA in a sample obtained from a subject; and (c) comparing the relative level, amount or activity obtained in step (a) with the level, amount or activity obtained in step (b).
 34. A method of claim 33, wherein the therapeutic is selected from the group consisting of: an agent that suppresses the development of a hyperplasia or an agent that directly inhibits cellular growth.
 35. A method of claim 34, wherein the agent that suppresses the development of a hyperplasia is selected from the group consisting of; a lipid lowering drug, antiplatelet agent, an anti-inflammatory agent, an antihypertensive agent and an anticoagulant.
 36. A method of claim 33, wherein the therapeutic is a modulator of an IL-1 activity.
 37. A method of claim 36, wherein the IL-1 activity is IL-1α.
 38. A method of claim 36, wherein the IL-1 activity is IL-1β.
 39. A method of claim 36, wherein the IL-1 activity is IL-1RN
 40. A method of claim 34, wherein the therapeutic is a protein, peptide, peptidomimetic, small molecule or a nucleic acid.
 41. A method of claim 36, wherein the modulator is an agonist.
 42. A method of claim 36, wherein the modulator is an antagonist.
 43. A method for treating or preventing the development of a restenosis in a subject comprising the steps of detecting the presence of a restenosis associated allele and administering to the subject a therapeutic that compensates for causative mutation that is in linkage disequilibrium with the restenosis associated allele.
 44. A method of claim 43, wherein the detecting step is selected from the group consisting of: a) allele specific oligonucleotide hybridization; b) size analysis; c) sequencing; d) hybridization; e) 5′ nuclease digestion; f) single-stranded conformation polymorphism; g) allele specific hybridization; h) primer specific extension; and j) oligonucleotide ligation assay.
 45. A method of claim 43, wherein prior to or in conjunction with detecting, the nucleic acid sample is subjected to an amplification step.
 46. A method of claim 45, wherein said amplification step employs a primer selected from the group consisting of any of SEQ ID Nos. 1-14.
 47. A method of claim 44, wherein said size analysis is preceded by a restriction enzyme digestion.
 48. A method of claim 47, wherein said restriction enzyme digestion uses a restriction enzyme selected from the group consisting of Alu I, Msp I, Nco I, Fnu 4HI, Ava I, Bsu 36 I, and Taq I.
 49. A method of claim 43, wherein the therapeutic is selected from the group consisting of: an agent that suppresses the development of a hyperplasia or an agent that directly inhibits cellular growth.
 50. A method of claim 49, wherein the agent that suppresses the development of a hyperplasia is selected from the group consisting of; a lipid lowering drug, antiplatelet agent, an anti-inflammatory agent, an antihypertensive agent and an anticoagulant.
 51. A method of claim 43, wherein the therapeutic is selected from the group consisting of: a modulator of an IL-1 activity.
 52. A method of claim 51, wherein the IL-1 activity is IL-1α.
 53. A method of claim 51, wherein the IL-1 activity is IL-1β.
 54. A method of claim 51, wherein the IL-1 activity is IL-1Ra.
 55. A method of claim 51, wherein the therapeutic is a protein, peptide, peptidomimetic, small molecule or a nucleic acid.
 56. A method of claim 51, wherein the modulator is an agonist.
 57. A method of claim 51, wherein the modulator is an antagonist.
 58. A method of claim 43, wherein the restenosis associated allele is allele 1 of any of the following markers: IL-1A (+4845), IL-1B (−511), IL-1B (+3954) and IL-1RN (+2018) or an allele in linkage disequilibrium therewith.
 59. A method of claim 43, wherein the ILD causative functional mutation is IL-1B (+6912) allele 2, IL-1B (−511) allele 2 or IL-1RN (+2018) allele
 2. 60. A method for screening for a restenosis therapeutic comprising the steps of: a) combining an IL-1 polypeptide or bioactive fragment thereof, an IL-1 binding partner and a test compound under conditions wherein, but for the test compound, the IL-1 protein and IL-1 binding partner are able to interact; and b) detecting the extent to which, in the presence of the test compound, an IL-1 protein/IL-1 binding partner complex is formed, wherein an increase in the amount of complex formed by an agonist in the presence of the compound relative to in the absence of the compound or a decrease in the amount of complex formed by an antagonist in the presence of the compound relative to in the absence of the compound indicates that the compound is a restenosis therapeutic.
 61. A method of claim 60, wherein the agonist or antagonist is selected from the group consisting of: a protein, peptide, peptidomimetic, small molecule or nucleic acid.
 62. A method of claim 61, wherein the nucleic acid is selected from the group consisting of: an antisense, ribozyme and triplex nucleic acid.
 63. A method of claim 60, which additionally comprises the step of preparing a pharmaceutical composition from the compound.
 64. A method of claim 60, wherein the IL-1 polypeptide is IL-1α.
 65. A method of claim 60, wherein the IL-1 polypeptide is IL-1β.
 66. A method of claim 60, wherein the IL-1 polypeptide is IL-1Ra.
 67. A method for identifying a restenosis therapeutic, comprising the steps of: a) contacting an appropriate amount of a candidate compound with a cell or cellular extract, which expresses an IL-1 gene; and b) determining the resulting protein bioactivity, wherein a decrease of an agonist bioactivity or a decrease in an antagonist bioactivity in the presence of the compound as compared to the bioactivity in the absence of the compound indicates that the candidate is a restenosis therapeutic.
 68. A method of claim 67, wherein the modulator is an antagonist of an IL-1α or an IL-1β, bioactivity.
 69. A method of claim 67, wherein the modulator is an agonist of an IL-1RN bioactivity.
 70. A method of claim 67, wherein in step (b), the protein bioactivity is determined by determining the expression level of an IL-1 gene.
 71. A method of claim 70, wherein the expression level is determined by detecting the amount of mRNA transcribed from an IL-1 gene.
 72. A method of claim 70, wherein the expression level is determined by detecting the amount of the IL-1 product produced.
 73. A method of claim 70, wherein the expression level is determined using an anti-the IL-1 antibody in an immunodetection assay.
 74. A method of claim 70, which additionally comprises the step of preparing a pharmaceutical composition from the compound.
 75. A method of claim 70, wherein said cell is contained in an animal.
 76. A method of claim 75, wherein the animal is transgenic.
 77. The method of any of claims 1, 16 or 43, wherein the presence of an IL-1 locus allelic pattern comprising allele 1 of each of IL-1A (+4845), IL-1B (+3954), IL-1B (−511), and IL-1RN (+2018), is detected.
 78. The method of claim 77, further comprising determining whether allele 1 of IL-1RN (+2018) is carried in the homozygous state.
 79. A method of claim 1, wherein the restenosis allele is selected from the group consisting of allele 1 of any of the following markers: IL-1A (+4845), IL-1B (−511), IL-1B (+3954) and IL-1RN (+2018) or an allele in linkage disequilibrium therewith. 